Compounds for enzyme inhibition

ABSTRACT

One aspect of the invention relates to inhibitors that preferentially inhibit immunoproteasome activity over constitutive proteasome activity. In certain embodiments, the invention relates to the treatment of immune related diseases, comprising administering a compound of the invention. In certain embodiments, the invention relates to the treatment of cancer, comprising administering a compound of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/078,378, filed Nov. 12, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/328,909, filed Apr. 12, 2012, which is acontinuation of U.S. patent application Ser. No. 12/708,753, filed Feb.19, 2010, issued as U.S. Pat. No. 8,080,545 on Dec. 20, 2011, which is acontinuation of U.S. patent application Ser. No. 11/820,490, filed Jun.19, 2007, issued as U.S. Pat. No. 7,691,852, which claims the benefit ofU.S. Provisional Application No. 60/815,218, filed Jun. 19, 2006. Thisapplication is related to U.S. patent application Ser. No. 12/708,932,filed Feb. 19, 2010, issued as U.S. Pat. No. 8,080,576. The contents ofthis application are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I presentation,apoptosis and cell viability, antigen processing, NF-κB activation, andtransduction of proinflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits, classified as α- and β-type,that are arranged in 4 stacked heptameric rings. In yeast and othereukaryotes, 7 different a subunits form the outer rings and 7 differentβ subunits comprise the inner rings. The a subunits serve as bindingsites for the 19S (PA700) and 11S (PA28) regulatory complexes, as wellas a physical barrier for the inner proteolytic chamber formed by thetwo β subunit rings. Thus, in vivo, the proteasome is believed to existas a 26S particle (“the 26S proteasome”). In vivo experiments have shownthat inhibition of the 20S form of the proteasome can be readilycorrelated to inhibition of 26S proteasome.

Cleavage of amino-terminal prosequences of β subunits during particleformation expose amino-terminal threonine residues, which serve as thecatalytic nucleophiles. The subunits responsible for catalytic activityin proteasome thus possess an amino terminal nucleophilic residue, andthese subunits belong to the family of N-terminal nucleophile (Ntn)hydrolases (where the nucleophilic N-terminal residue is, for example,Cys, Ser, Thr, and other nucleophilic moieties). This family includes,for example, penicillin G acylase (PGA), penicillin V acylase (PVA),glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three interferon-γ-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, β5, β1 and β2, respectively. When all threeIFN-γ-inducible subunits are present, the proteasome is referred to asan “immunoproteasome”. Thus, eukaryotic cells can possess two forms ofproteasomes in varying ratios.

Through the use of different peptide substrates, three major proteolyticactivities have been defined for the eukaryote 20S proteasomes:chymotrypsin-like activity (CT-L), which cleaves after large hydrophobicresidues; trypsin-like activity (T-L), which cleaves after basicresidues; and peptidylglutamyl peptide hydrolyzing activity (PGPH),which cleaves after acidic residues. Two additional less characterizedactivities have also been ascribed to the proteasome: BrAAP activity,which cleaves after branched-chain amino acids; and SNAAP activity,which cleaves after small neutral amino acids. Although both forms ofthe proteasome possess all five enzymatic activities, differences in theextent of the activities between the forms have been described based onspecific substrates. For both forms of the proteasome, the majorproteasome proteolytic activities appear to be contributed by differentcatalytic sites within the 20S core.

There are several examples of small molecules which have been used toinhibit proteasome activity; however, these compounds generally lack thespecificity to delineate between the two forms of the proteasome. Thus,the ability to explore and exploit the roles of each specific proteasomeform at the cellular and molecular level has not been possible.Therefore, the creation of small molecule inhibitor(s) thatpreferentially inhibit a single form of the proteasome is needed toallow the exploration of the roles of each proteasome form at thecellular and molecular level.

SUMMARY OF THE INVENTION

One aspect of the invention relates to inhibitors that preferentiallyinhibit immunoproteasome activity over constitutive proteasome activity.In certain embodiments, the invention relates to the treatment of immunerelated diseases, comprising administering a compound of the invention.In certain embodiments, the invention relates to the treatment ofcancer, comprising administering a compound of the invention.

One aspect of the invention relates to compounds having a structure offormula (I) or a pharmaceutically acceptable salt thereof,

wherein

each Ar is independently an aromatic or heteroaromatic group optionallysubstituted with 1 to 4 substituents;

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

X is selected from O, S, NH, and N—C₁₋₆alkyl, preferably O;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl,aryl, and C₁₋₆aralkyl;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, andC₁₋₆aralkyl, preferably hydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

R¹⁵ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,—C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

Another aspect of the invention relates to compounds having a structureof formula (II) or a pharmaceutically acceptable salt thereof,

each Ar is independently an aromatic or heteroaromatic group optionallysubstituted with 1 to 4 substituents;

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

X is selected from O, S, NH, and N—C₁₋₆alkyl, preferably O;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈ alkyl-, (R¹³)₂N—C₁₋₁₂ alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁸ is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl, preferablyhydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

R¹⁵ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,—C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

Another aspect of the invention relates to compounds having a structureof formula (III) or a pharmaceutically acceptable salt thereof,

wherein

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

W is selected from —CHO and —B(OR¹¹)₂;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl,aryl, and C₁₋₆aralkyl;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, andC₁₋₆aralkyl, preferably hydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

each R¹⁶ is independently selected from hydrogen and C₁₋₆alkyl; or twooccurrences of R¹¹ together may be C₁₋₆alkyl, thereby forming a ringtogether with the intervening boron and oxygen atoms to which they areattached;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

Another aspect of the invention relates to compounds having a structureof formula (IV) or a pharmaceutically acceptable salt thereof,

wherein

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

W is selected from —CHO and —B(OR¹¹)₂;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁸ is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl, preferablyhydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

each R¹⁶ is independently selected from hydrogen and C₁₋₆alkyl; or twooccurrences of R¹¹ together may be C₁₋₆alkyl, thereby forming a ringtogether with the intervening boron and oxygen atoms to which they areattached;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the immunoproteasome expression level of certain cell linesand patient samples, including multiple myeloma, leukemias, lymphomas,and solid tumors.

FIG. 2A shows the effect of Compound 14 on disease progression in mousemodels of rheumatoid arthritis (RA) where dosing began when animalsfirst showed signs of disease (indicated by arrows) and data shown isaverage disease score (±SEM; N=7/group) and is representative of threeindependent experiments.

FIG. 2B shows the effect of Compound 14 on disease progression in mousemodels of RA where RA was induced on Day 0 in female DBA/1 mice byimmunization with bovine type II collagen in CFA where dosing began whenanimals first showed signs of disease (indicated by arrows) and datashown is average disease score (±SEM; N=10/group).

DETAILED DESCRIPTION OF THE INVENTION

The invention involves compounds useful as enzyme inhibitors. Thesecompounds are generally useful to inhibit enzymes having a nucleophilicgroup at the N-terminus. For example, activities of enzymes or enzymesubunits having N-terminal amino acids with nucleophiles in their sidechains, such as threonine, serine, or cysteine can be successfullyinhibited by the enzyme inhibitors described herein. Activities ofenzymes or enzyme subunits having non-amino acid nucleophilic groups attheir N-termini, such as, for example, protecting groups orcarbohydrates, can also be successfully inhibited by the enzymeinhibitors described herein.

While not bound by any particular theory of operation, it is believedthat such N-terminal nucleophiles of Ntn form covalent adducts with theepoxide, aziridine, aldehyde, or borate functional group of the enzymeinhibitors described herein. For example, in the β5/Pre2 subunit of 20Sproteasome, the N-terminal threonine is believed to irreversibly form amorpholino or piperazino adduct upon reaction with a peptide epoxide oraziridine such as those described below. Such adduct formation wouldinvolve ring-opening cleavage of the epoxide or aziridine.

Regarding the stereochemistry, the Cahn-Ingold-Prelog rules fordetermining absolute stereochemistry are followed. These rules aredescribed, for example, in Organic Chemistry, Fox and Whitesell; Jonesand Bartlett Publishers, Boston, Mass. (1994); Section 5-6, pp 177-178,which section is hereby incorporated by reference. Peptides can have arepeating backbone structure with side chains extending from thebackbone units. Generally, each backbone unit has a side chainassociated with it, although in some cases, the side chain is a hydrogenatom. In other embodiments, not every backbone unit has an associatedside chain. Peptides useful in peptide epoxides or peptide aziridineshave two or more backbone units. In some embodiments useful forinhibiting chymotrypsin-like (CT-L) activity of the proteasome, betweentwo and eight backbone units are present, and in some preferredembodiments for CT-L inhibition, between two and six backbone units arepresent.

The side chains extending from the backbone units can include naturalaliphatic or aromatic amino acid side chains, such as hydrogen(glycine), methyl (alanine), isopropyl (valine), sec-butyl (isoleucine),isobutyl (leucine), phenylmethyl (phenylalanine), and the side chainconstituting the amino acid proline. The side chains can also be otherbranched or unbranched aliphatic or aromatic groups such as ethyl,n-propyl, n-butyl, t-butyl, and aryl substituted derivatives such as1-phenylethyl, 2-phenylethyl, (1-naphthyl)methyl, (2-naphthyl)methyl,1-(1-naphthyl)ethyl, 1-(2-naphthyl)ethyl, 2-(1-naphthyl)ethyl,2-(2-naphthyl)ethyl, and similar compounds. The aryl groups can befurther substituted with branched or unbranched C1-6alkyl groups, orsubstituted alkyl groups, acetyl and the like, or further aryl groups,or substituted aryl groups, such as benzoyl and the like. Heteroarylgroups can also be used as side chain substituents. Heteroaryl groupsinclude nitrogen-, oxygen-, and sulfur-containing aryl groups such asthienyl, benzothienyl, naphthothienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl,pyrazinyl, indolyl, purinyl, quinolyl, and the like.

In some embodiments, polar or charged residues can be introduced intothe peptide epoxides or peptide aziridines. For example, naturallyoccurring amino acids such as hydroxy-containing (Thr, Tyr, Ser) orsulfur-containing (Met, Cys) can be introduced, as well as non-essentialamino acids, for example, taurine, carnitine, citrulline, cystine,ornithine, norleucine and others. Non-naturally occurring side chainsubstituents with charged or polar moieties can also be included, suchas, for example, C1-6alkyl chains or C6-12aryl groups with one or morehydroxy, short chain alkoxy, sulfide, thio, carboxyl, ester, phospho,amido or amino groups, or such substituents substituted with one or morehalogen atoms. In some preferred embodiments, there is at least one arylgroup present in a side chain of the peptide moiety.

In some embodiments, the backbone units are amide units[—NH—CHR—C(═O)—], in which R is the side chain. Such a designation doesnot exclude the naturally occurring amino acid proline, or othernon-naturally occurring cyclic secondary amino acids, which will berecognized by those of skill in the art.

In other embodiments, the backbone units are N-alkylated amide units(for example, N-methyl and the like), olefinic analogs (in which one ormore amide bonds are replaced by olefinic bonds), tetrazole analogs (inwhich a tetrazole ring imposes a cis-configuration on the backbone), orcombinations of such backbone linkages. In still other embodiments, theamino acid α-carbon is modified by α-alkyl substitution, for example,aminoisobutyric acid. In some further embodiments, side chains arelocally modified, for example, by ΔE or ΔZ dehydro modification, inwhich a double bond is present between the α and β atoms of the sidechain, or for example by ΔE or ΔZ cyclopropyl modification, in which acyclopropyl group is present between the α and β atoms of the sidechain. In still further embodiments employing amino acid groups, D-aminoacids can be used. Further embodiments can include sidechain-to-backbone cyclization, disulfide bond formation, lactamformation, azo linkage, and other modifications discussed in “Peptidesand Mimics, Design of Conformationally Constrained” by Hruby and Boteju,in “Molecular Biology and Biotechnology: A Comprehensive DeskReference”, ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664,which is hereby incorporated by reference.

One aspect of the invention relates to compounds having a structure offormula (I) or a pharmaceutically acceptable salt thereof,

wherein

each Ar is independently an aromatic or heteroaromatic group optionallysubstituted with 1 to 4 substituents;

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

X is selected from O, S, NH, and N—C₁₋₆alkyl, preferably O;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl,aryl, and C₁₋₆aralkyl;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group, more preferablyt-butoxycarbonyl or benzyloxycarbonyl; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, andC₁₋₆aralkyl, preferably hydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

R¹⁵ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,—C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl, preferably C₁₋₆alkyland C₁₋₆hydroxyalkyl, more preferably methyl, ethyl, hydroxymethyl, and2-hydroxyethyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R¹ is selected from —C₁₋₆alkyl-B andC₁₋₆aralkyl. In certain such embodiments, R¹ is substituted with one ormore substituents selected from hydroxy, halogen, amide, amine,carboxylic acid (or a salt thereof), ester (including C₁₋₆alkyl ester,C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainpreferred such embodiments, R¹ is substituted with one or moresubstituents selected from carboxylic acid and ester. In certainembodiments, R¹ is selected from methyl, ethyl, isopropyl,carboxymethyl, and benzyl. In certain embodiments R¹ is —C₁₋₆alkyl-B andC₁₋₆aralkyl. In certain preferred such embodiments, B is absent.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, wherein the alkylmoiety may contain six, five, four, three, two, or one carbon atoms,preferably one or two. In certain such embodiments, R² is substitutedwith one or more substituents selected from hydroxy, halogen, amide,amine, carboxylic acid (or a salt thereof), ester (including C₁₋₆alkylester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainsuch embodiments, R² is substituted with a substituent selected fromalkyl, trihaloalkyl, alkoxy, hydroxy, or cyano. In certain suchembodiments, R² is selected from C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl.In certain preferred such embodiments, R² is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ and CH₃. Incertain embodiments D is selected from H, OMe, OH, CN, CF₃ and CH₃.

In certain preferred such embodiments where D is attached to asix-membered ring, D is attached at the 4-position relative to the pointof attachment, preferably excluding embodiments where the 4-position ofthe ring is occupied by the nitrogen of a pyridine ring.

In certain embodiments, R³ is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl, wherein the alkyl moiety may contain six, five, four,three, two, or one carbon atoms, preferably one or two. In certain suchembodiments, R³ is substituted with one or more substituents selectedfrom hydroxy, halogen, amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether. In certain such embodiments, R³ issubstituted with a substituent selected from alkyl, trihaloalkyl,alkoxy, hydroxy, or cyano. In certain such embodiments, R³ is selectedfrom C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl. In certain preferred suchembodiments, R³ is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ or CH₃. Incertain embodiments, D is selected from H, OMe, OH, CN, CF₃ or CH₃.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-E-, where E is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Q is C₁₋₆alkyl, Q maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Q is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Q is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, L and Q are absent and R⁶ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁵ is C₁₋₆alkyl and R⁶ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁶ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁶ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁶ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹³)₂N—C₁₋₈alkyl-, (R¹³)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R⁶ is H.

In certain embodiments, R⁵ is C₁₋₆alkyl, R⁶ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁶ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁶ is C₁₋₆aralkyl. Incertain such embodiments, R⁶ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁵ is C₁₋₆alkyl, and R⁶ isaryl. In certain such embodiments, R⁶ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheteroaryl and C₁₋₆heteroaralkyl. In certain such embodiments, R⁶ isheteroaryl selected from pyrrole, furan, thiophene, imidazole,isoxazole, oxazole, oxadiazole, thiazole, thiadiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. In certainalternative such embodiments, R⁶ is C₁₋₆heteroaralkyl selected frompyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl,isoxazolylmethyl, oxazolylmethyl, oxadiazolylmethyl, thiazolylmethyl,thiadiazolylmethyl, triazolylmethyl, pyrazolylmethyl, pyridylmethyl,pyrazinylmethyl, pyridazinylmethyl and pyrimidinylmethyl.

In certain embodiments, L is C═O, Q is absent or O, and R⁶ iscarbocyclylM-, wherein M is C₀₋₁alkyl. In certain such embodiments, R⁶is cyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, M isC₁₋₈alkyl, preferably methylene, and R⁶ is selected fromR¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-, R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁶ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹⁶)(R¹⁷), wherein R¹⁶ and R¹⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, M is C₁₋₈alkyl,and R⁶ is selected from (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂NC₁₋₈alkyl,(R¹³)₃N⁺C₁₋₈alkyl-, and heterocyclyl-M-. In certain such embodiments, R⁶is (R¹³)₂NC₁₋₈alkyl or (R¹³)₃N⁺C₁₋₈alkyl-, where R¹³ is C₁₋₆alkyl. Incertain other such embodiments, R⁶ is heterocyclylM-, where heterocyclylis selected from morpholino, piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q is selected from Oand NH and R⁶ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q isselected from O and NH, and R⁶ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁵is C₁₋₆alkyl, Q is selected from O and NH and R⁶ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁵ isC₁₋₆alkyl, Q is selected from O and NH, and R⁶ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁵ and R⁶ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁵ and R⁶ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁵ and R⁶together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁵ and R⁶ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁵ and R⁶ together areC₂₋₃alkyl-A.

In certain embodiments, R⁷ and R⁸ are independently selected fromhydrogen and C₁₋₆alkyl. In certain preferred such embodiments, R⁷ and R⁸are independently selected from hydrogen and methyl. In more preferredsuch embodiments, R⁷ and R⁸ are both hydrogen.

In certain embodiments, X is O, R² and R³ are each independentlyC₁₋₆aralkyl, and R¹ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of which is optionallysubstituted with one or more of amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether substituents.

Suitable N-terminal protecting groups known in the art of peptidesyntheses, include t-butoxy carbonyl (Boc), benzoyl (Bz),fluoren-9-ylmethoxycarbonyl (Fmoc), triphenylmethyl (trityl) andtrichloroethoxycarbonyl (Troc) and the like. The use of variousN-protecting groups, e.g., the benzyloxy carbonyl group or thet-butyloxycarbonyl group (Boc), various coupling reagents, e.g.,dicyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),N-hydroxyazabenzotriazole (HATU), carbonyldiimidazole, or1-hydroxybenzotriazole monohydrate (HOBT), and various cleavageconditions: for example, trifluoracetic acid (TFA), HCl in dioxane,hydrogenation on Pd—C in organic solvents (such as methanol or ethylacetate), boron tris(trifluoroacetate), and cyanogen bromide, andreaction in solution with isolation and purification of intermediatesare well-known in the art of peptide synthesis, and are equallyapplicable to the preparation of the subject compounds. Suitableprotecting N-terminal protecting groups may also be found, for example,in Greene, T. W.; Wuts, P. G. M. “Protective Groups in OrganicSynthesis”, 3rd ed.; Wiley: New York, 1999 or Kocieński, P. J.,“Protecting Groups”, Georg Thieme Verlag, 1994.

In certain embodiments, the stereochemical configuration of the carbonsbearing R¹, R², or R³ are independently D or L. In certain preferredembodiments, the stereochemical configuration of at least one of thecarbons bearing R¹, R², and R³ respectively is D. In certain preferredsuch embodiments, the stereochemical configuration of the carbon bearingR¹ is D. In certain such embodiments, the stereochemical configurationof the carbon bearing R² is D. In certain such embodiments, thestereochemical configuration of the carbon bearing R³ is D. In certainembodiments the stereochemical configuration of at least two of thecarbons bearing R¹, R², and R³ respectively is D. In yet anotherpreferred embodiment, the stereochemical configuration of all three ofthe carbons bearing R¹, R², and R³ respectively is D.

Another aspect of the invention relates to compounds having a structureof formula (II) or a pharmaceutically acceptable salt thereof,

each Ar is independently an aromatic or heteroaromatic group optionallysubstituted with 1 to 4 substituents;

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

X is selected from O, S, NH, and N—C₁₋₆alkyl, preferably O;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group, more preferablyt-butoxycarbonyl or benzyloxycarbonyl; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁸ is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl, preferablyhydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

R¹⁵ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,—C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl, preferably C₁₋₆alkyland C₁₋₆hydroxyalkyl, more preferably methyl, ethyl, hydroxymethyl, and2-hydroxyethyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, wherein the alkylmoiety may contain six, five, four, three, two, or one carbon atoms,preferably one or two. In certain such embodiments, R² is substitutedwith one or more substituents selected from hydroxy, halogen, amide,amine, carboxylic acid (or a salt thereof), ester (including C₁₋₆alkylester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainsuch embodiments, R² is substituted with a substituent selected fromalkyl, trihaloalkyl, alkoxy, hydroxy, or cyano. In certain suchembodiments, R² is selected from C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl.In certain preferred such embodiments, R² is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ and CH₃. Incertain embodiments D is selected from H, OMe, OH, CN, CF₃ and CH₃.

In certain preferred such embodiments where D is attached to asix-membered ring, D is attached at the 4-position relative to the pointof attachment, preferably excluding embodiments where the 4-position ofthe ring is occupied by the nitrogen of a pyridine ring.

In certain embodiments, R³ is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain the alkyl moiety may contain six, five,four, three, two, or one carbon atoms, preferably one or two. In certainsuch embodiments, R³ is substituted with one or more substituentsselected from hydroxy, halogen, amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether. In certain such embodiments, R³ issubstituted with a substituent selected from alkyl, trihaloalkyl,alkoxy, hydroxy, or cyano. In certain such embodiments, R³ is selectedfrom C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl. In certain preferred suchembodiments, R³ is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ or CH₃. Incertain embodiments, D is selected from H, OMe, OH, CN, CF₃ or CH₃.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-E-, where E is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Q is C₁₋₆alkyl, Q maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Q is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Q is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, L and Q are absent and R⁶ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁵ is C₁₋₆alkyl and R⁶ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁶ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁶ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁶ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹³)₂N—C₁₋₈alkyl-, (R¹³)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R⁶ is H.

In certain embodiments, R⁵ is C₁₋₆alkyl, R⁶ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁶ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁶ is C₁₋₆aralkyl. Incertain such embodiments, R⁶ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁵ is C₁₋₆alkyl, and R⁶ isaryl. In certain such embodiments, R⁶ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheteroaryl and C₁₋₆heteroaralkyl. In certain such embodiments, R⁶ isheteroaryl selected from pyrrole, furan, thiophene, imidazole,isoxazole, oxazole, oxadiazole, thiazole, thiadiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. In certainalternative such embodiments, R⁶ is C₁₋₆heteroaralkyl selected frompyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl,isoxazolylmethyl, oxazolylmethyl, oxadiazolylmethyl, thiazolylmethyl,thiadiazolylmethyl, triazolylmethyl, pyrazolylmethyl, pyridylmethyl,pyrazinylmethyl, pyridazinylmethyl and pyrimidinylmethyl.

In certain embodiments, L is C═O, Q is absent or O, and R⁶ iscarbocyclylM-, wherein M is C₀₋₁alkyl. In certain such embodiments, R⁶is cyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, M isC₁₋₈alkyl, preferably methylene, and R⁶ is selected fromR¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-, R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁶ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹⁶)(R¹⁷), wherein R¹⁶ and R¹⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, M is C₁₋₈alkyl,and R⁶ is selected from (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂NC₁₋₈alkyl,(R¹³)₃N⁺C₁₋₈alkyl-, and heterocyclyl-M-. In certain such embodiments, R⁶is (R¹³)₂NC₁₋₈alkyl or (R¹³)₃N⁺C₁₋₈alkyl-, where R¹³ is C₁₋₆alkyl. Incertain other such embodiments, R⁶ is heterocyclylM-, where heterocyclylis selected from morpholino, piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q is selected from Oand NH and R⁶ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q isselected from O and NH, and R⁶ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁵is C₁₋₆alkyl, Q is selected from O and NH and R⁶ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁵ isC₁₋₆alkyl, Q is selected from O and NH, and R⁶ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁵ and R⁶ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁵ and R⁶ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁵ and R⁶together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁵ and R⁶ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁵ and R⁶ together areC₂₋₃alkyl-A.

In certain embodiments, R⁸ is selected from hydrogen and C₁₋₆alkyl. Incertain preferred such embodiments, R⁸ is selected from hydrogen andmethyl. In more preferred such embodiments, R⁸ is hydrogen.

In certain embodiments, X is O, R² and R³ are each independentlyC₁₋₆aralkyl, and R¹ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of which is optionallysubstituted with one or more of amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether substituents.

In certain embodiments, the stereochemical configuration of the carbonsbearing R² or R³ are independently D or L. In certain preferredembodiments, the stereochemical configuration of at least one of thecarbons bearing R² and R³ respectively is D. In certain suchembodiments, the stereochemical configuration of the carbon bearing R²is D. In such embodiments, the stereochemical configuration of thecarbon bearing R³ is D. In certain embodiments, the stereochemicalconfiguration of both of the carbons bearing R² and R³ respectively isD.

Another aspect of the invention relates to compounds having a structureof formula (III) or a pharmaceutically acceptable salt thereof,

wherein

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

W is selected from —CHO and —B(OR¹¹)₂;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl,aryl, and C₁₋₆aralkyl;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group, more preferablyt-butoxycarbonyl or benzyloxycarbonyl; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, andC₁₋₆aralkyl, preferably hydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

each R¹⁶ is independently selected from hydrogen and C₁₋₆alkyl; or twooccurrences of R¹¹ together may be C₁₋₆alkyl, thereby forming a ringtogether with the intervening boron and oxygen atoms to which they areattached;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R¹ is selected from —C₁₋₆alkyl-B andC₁₋₆aralkyl. In certain such embodiments, R¹ is substituted with one ormore substituents selected from hydroxy, halogen, amide, amine,carboxylic acid (or a salt thereof), ester (including C₁₋₆alkyl ester,C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainpreferred such embodiments, R¹ is substituted with one or moresubstituents selected from carboxylic acid and ester. In certainembodiments, R¹ is selected from methyl, ethyl, isopropyl,carboxymethyl, and benzyl. In certain embodiments R¹ is —C₁₋₆alkyl-B andC₁₋₆aralkyl. In certain preferred such embodiments, B is absent.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, wherein the alkylmoiety may contain six, five, four, three, two, or one carbon atoms,preferably one or two. In certain such embodiments, R² is substitutedwith one or more substituents selected from hydroxy, halogen, amide,amine, carboxylic acid (or a salt thereof), ester (including C₁₋₆alkylester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainsuch embodiments, R² is substituted with a substituent selected fromalkyl, trihaloalkyl, alkoxy, hydroxy, or cyano. In certain suchembodiments, R² is selected from C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl.In certain preferred such embodiments, R² is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ and CH₃. Incertain embodiments D is selected from H, OMe, OH, CN, CF₃ and CH₃.

In certain preferred such embodiments where D is attached to asix-membered ring, D is attached at the 4-position relative to the pointof attachment, preferably excluding embodiments where the 4-position ofthe ring is occupied by the nitrogen of a pyridine ring.

In certain embodiments, R³ is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain the alkyl moiety may contain six, five,four, three, two, or one carbon atoms, preferably one or two. In certainsuch embodiments, R³ is substituted with one or more substituentsselected from hydroxy, halogen, amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether. In certain such embodiments, R³ issubstituted with a substituent selected from alkyl, trihaloalkyl,alkoxy, hydroxy, or cyano. In certain such embodiments, R³ is selectedfrom C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl. In certain preferred suchembodiments, R³ is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ or CH₃. Incertain embodiments, D is selected from H, OMe, OH, CN, CF₃ or CH₃.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-E-, where E is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Q is C₁₋₆alkyl, Q maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Q is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Q is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, L and Q are absent and R⁶ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁵ is C₁₋₆alkyl and R⁶ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁶ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁶ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁶ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹³)₂N—C₁₋₈alkyl-, (R¹³)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R⁶ is H.

In certain embodiments, R⁵ is C₁₋₆alkyl, R⁶ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁶ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁶ is C₁₋₆aralkyl. Incertain such embodiments, R⁶ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁵ is C₁₋₆alkyl, and R⁶ isaryl. In certain such embodiments, R⁶ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheteroaryl and C₁₋₆heteroaralkyl. In certain such embodiments, R⁶ isheteroaryl selected from pyrrole, furan, thiophene, imidazole,isoxazole, oxazole, oxadiazole, thiazole, thiadiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. In certainalternative such embodiments, R⁶ is C₁₋₆heteroaralkyl selected frompyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl,isoxazolylmethyl, oxazolylmethyl, oxadiazolylmethyl, thiazolylmethyl,thiadiazolylmethyl, triazolylmethyl, pyrazolylmethyl, pyridylmethyl,pyrazinylmethyl, pyridazinylmethyl and pyrimidinylmethyl.

In certain embodiments, L is C═O, Q is absent or O, and R⁶ iscarbocyclylM-, wherein M is C₀₋₁alkyl. In certain such embodiments, R⁶is cyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, M isC₁₋₈alkyl, preferably methylene, and R⁶ is selected fromR¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-, R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁶ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹⁶)(R¹⁷), wherein R¹⁶ and R¹⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, M is C₁₋₈alkyl,and R⁶ is selected from (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂NC₁₋₈alkyl,(R¹³)₃N⁺C₁₋₈alkyl-, and heterocyclyl-M-. In certain such embodiments, R⁶is (R¹³)₂NC₁₋₈alkyl or (R¹³)₃N⁺C₁₋₈alkyl-, where R¹³ is C₁₋₆alkyl. Incertain other such embodiments, R⁶ is heterocyclylM-, where heterocyclylis selected from morpholino, piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q is selected from Oand NH and R⁶ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q isselected from O and NH, and R⁶ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁵is C₁₋₆alkyl, Q is selected from O and NH and R⁶ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁵ isC₁₋₆alkyl, Q is selected from O and NH, and R⁶ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁵ and R⁶ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁵ and R⁶ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁵ and R⁶together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁵ and R⁶ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁵ and R⁶ together areC₂₋₃alkyl-A.

In certain embodiments, R⁷ and R⁸ are independently selected fromhydrogen and C₁₋₆alkyl. In certain preferred such embodiments, R⁷ and R⁸are independently selected from hydrogen and methyl. In more preferredsuch embodiments, R⁷ and R⁸ are both hydrogen.

In certain embodiments, X is O, R² and R³ are each independentlyC₁₋₆aralkyl, and R¹ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of which is optionallysubstituted with one or more of amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether substituents.

In certain embodiments, the stereochemical configuration of the carbonsbearing R¹, R², or R³ are independently D or L. In certain preferredembodiments, the stereochemical configuration of at least one of thecarbons bearing R¹, R², and R³ respectively is D. In certain preferredsuch embodiments, the stereochemical configuration of the carbon bearingR¹ is D. In certain such embodiments, the stereochemical configurationof the carbon bearing R² is D. In certain such embodiments, thestereochemical configuration of the carbon bearing R³ is D. In certainembodiments the stereochemical configuration of at least two of thecarbons bearing R¹, R², and R³ respectively is D. In yet anotherpreferred embodiment, the stereochemical configuration of all three ofthe carbons bearing R¹, R², and R³ respectively is D.

Another aspect of the invention relates to compounds having a structureof formula (IV) or a pharmaceutically acceptable salt thereof,

wherein

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

W is selected from —CHO and —B(OR¹¹)₂;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,heteroaryl, and C₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group, more preferablyt-butoxycarbonyl or benzyloxycarbonyl; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁸ is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl, preferablyhydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

each R¹⁶ is independently selected from hydrogen and C₁₋₆alkyl; or twooccurrences of R¹¹ together may be C₁₋₆alkyl, thereby forming a ringtogether with the intervening boron and oxygen atoms to which they areattached;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, wherein the alkylmoiety may contain six, five, four, three, two, or one carbon atoms,preferably one or two. In certain such embodiments, R² is substitutedwith one or more substituents selected from hydroxy, halogen, amide,amine, carboxylic acid (or a salt thereof), ester (including C₁₋₆alkylester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainsuch embodiments, R² is substituted with a substituent selected fromalkyl, trihaloalkyl, alkoxy, hydroxy, or cyano. In certain suchembodiments, R² is selected from C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl.In certain preferred such embodiments, R² is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ and CH₃. Incertain embodiments D is selected from H, OMe, OH, CN, CF₃ and CH₃.

In certain preferred such embodiments where D is attached to asix-membered ring, D is attached at the 4-position relative to the pointof attachment, preferably excluding embodiments where the 4-position ofthe ring is occupied by the nitrogen of a pyridine ring.

In certain embodiments, R³ is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain the alkyl moiety may contain six, five,four, three, two, or one carbon atoms, preferably one or two. In certainsuch embodiments, R³ is substituted with one or more substituentsselected from hydroxy, halogen, amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether. In certain such embodiments, R³ issubstituted with a substituent selected from alkyl, trihaloalkyl,alkoxy, hydroxy, or cyano. In certain such embodiments, R³ is selectedfrom C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl. In certain preferred suchembodiments, R³ is selected from

wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ or CH₃. Incertain embodiments, D is selected from H, OMe, OH, CN, CF₃ or CH₃.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-E-, where E is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Q is C₁₋₆alkyl, Q maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Q is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Q is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, L and Q are absent and R⁶ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁵ is C₁₋₆alkyl and R⁶ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁶ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁶ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁶ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹³)₂N—C₁₋₈alkyl-, (R¹³)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R⁶ is H.

In certain embodiments, R⁵ is C₁₋₆alkyl, R⁶ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁶ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁶ is C₁₋₆aralkyl. Incertain such embodiments, R⁶ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁵ is C₁₋₆alkyl, and R⁶ isaryl. In certain such embodiments, R⁶ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheteroaryl and C₁₋₆heteroaralkyl. In certain such embodiments, R⁶ isheteroaryl selected from pyrrole, furan, thiophene, imidazole,isoxazole, oxazole, oxadiazole, thiazole, thiadiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. In certainalternative such embodiments, R⁶ is C₁₋₆heteroaralkyl selected frompyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl,isoxazolylmethyl, oxazolylmethyl, oxadiazolylmethyl, thiazolylmethyl,thiadiazolylmethyl, triazolylmethyl, pyrazolylmethyl, pyridylmethyl,pyrazinylmethyl, pyridazinylmethyl and pyrimidinylmethyl.

In certain embodiments, L is C═O, Q is absent or O, and R⁶ iscarbocyclylM-, wherein M is C₀₋₁alkyl. In certain such embodiments, R⁶is cyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, M isC₁₋₈alkyl, preferably methylene, and R⁶ is selected fromR¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-, R¹¹ZA C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁶ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹⁶)(R¹⁷), wherein R¹⁶ and R¹⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, M is C₁₋₈alkyl,and R⁶ is selected from (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂NC₁₋₈alkyl,(R¹³)₃N⁺C₁₋₈alkyl-, and heterocyclyl-M-. In certain such embodiments, R⁶is (R¹³)₂NC₁₋₈alkyl or (R¹³)₃N⁺C₁₋₈alkyl-, where R¹³ is C₁₋₆alkyl. Incertain other such embodiments, R⁶ is heterocyclylM-, where heterocyclylis selected from morpholino, piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q is selected from Oand NH and R⁶ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q isselected from O and NH, and R⁶ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁵is C₁₋₆alkyl, Q is selected from O and NH and R⁶ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁵ isC₁₋₆alkyl, Q is selected from O and NH, and R⁶ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁵ and R⁶ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁵ and R⁶ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁵ and R⁶together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁵ and R⁶ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁵ and R⁶ together areC₂₋₃alkyl-A.

In certain embodiments, R⁸ is selected from hydrogen and C₁₋₆alkyl. Incertain preferred such embodiments, R⁸ is selected from hydrogen andmethyl. In more preferred such embodiments, R⁸ is hydrogen.

In certain embodiments, X is O, R² and R³ are each independentlyC₁₋₆aralkyl, and R¹ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of which is optionallysubstituted with one or more of amide, amine, carboxylic acid (or a saltthereof), ester (including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and arylester), thiol, or thioether substituents.

In certain embodiments, the stereochemical configuration of the carbonsbearing R² or R³ are independently D or L. In certain preferredembodiments, the stereochemical configuration of at least one of thecarbons bearing R² and R³ respectively is D. In certain suchembodiments, the stereochemical configuration of the carbon bearing R²is D. In such embodiments, the stereochemical configuration of thecarbon bearing R³ is D. In certain embodiments, the stereochemicalconfiguration of both of the carbons bearing R² and R³ respectively isD.

One aspect of the invention relates to inhibitors that preferentiallyinhibit immunoproteasome activity over constitutive proteasome activity.In certain embodiments, the EC₅₀ ratio of a compound of any one offormulae I to IV in an assay of constitutive proteasome activity ascompared to the EC₅₀ of the same compound in an assay ofimmunoproteasome activity is greater than 1. In certain suchembodiments, the EC₅₀ is greater than 2, 3, 4 or even 5. Suitable assaysfor the determination of the constitutive proteasome activity and theimmunoproteasome activity are described herein (see Example 18).

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, an amino group is basic,meaning it has a pK_(a)>7.00. The protonated forms of these functionalgroups have pK_(a)s above 7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

As used herein, “enzyme” can be any partially or wholly proteinaceousmolecule which carries out a chemical reaction in a catalytic manner.Such enzymes can be native enzymes, fusion enzymes, proenzymes,apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinatedenzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linkedenzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurringor artificially-generated mutant enzymes, enzymes with side chain orbackbone modifications, enzymes having leader sequences, and enzymescomplexed with non-proteinaceous material, such as proteoglycans,proteoliposomes. Enzymes can be made by any means, including naturalexpression, promoted expression, cloning, various solution-based andsolid-based peptide syntheses, and similar methods known to those ofskill in the art.

The term “C1-6heteroaralkyl”, as used herein, refers to a C1-6alkylgroup substituted with a heteroaryl group.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole,oxadiazole, thiazole, thiadiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example,tetrahydropyran, piperidine, piperazine, pyrrolidine, morpholine,lactones, lactams, and the like.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

As used herein, the term “inhibitor” is meant to describe a compoundthat blocks or reduces an activity of an enzyme (for example, inhibitionof proteolytic cleavage of standard fluorogenic peptide substrates suchas suc-LLVY-AMC, Box-LLR-AMC and Z-LLE-AMC, inhibition of variouscatalytic activities of the 20S proteasome). An inhibitor can act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitorcan bind reversibly or irreversibly, and therefore the term includescompounds that are suicide substrates of an enzyme. An inhibitor canmodify one or more sites on or near the active site of the enzyme, or itcan cause a conformational change elsewhere on the enzyme.

As used herein, the term “peptide” includes not only standard amidelinkage with standard α-substituents, but commonly utilizedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, as detailed below.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” encompasses compounds that, under physiologicalconditions, are converted into therapeutically active agents. A commonmethod for making a prodrug is to include selected moieties that arehydrolyzed under physiological conditions to reveal the desiredmolecule. In other embodiments, the prodrug is converted by an enzymaticactivity of the host animal.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S— alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

Uses of Enzyme Inhibitors

The biological consequences of proteasome inhibition are numerous.Proteasome inhibition has been suggested as a prevention and/ortreatment of a multitude of diseases including, but not limited to,proliferative diseases, neurotoxic/degenerative diseases, ischemicconditions, inflammation, immune-related diseases, HIV, cancers, organgraft rejection, septic shock, viral and parasitic infections,conditions associated with acidosis, macular degeneration, pulmonaryconditions, muscle wasting diseases, fibrotic diseases, bone and hairgrowth diseases.

Proteasome inhibitors can be used to treat conditions mediated directlyby the proteolytic function of the proteasome such as muscle wasting, ormediated indirectly via proteins which are processed by the proteasomesuch as NF-κB. The proteasome participates in the rapid elimination andpost-translational processing of proteins (e.g., enzymes) involved incellular regulation (e.g., cell cycle, gene transcription, and metabolicpathways), intercellular communication, and the immune response (e.g.,antigen presentation).

At the cellular level, the accumulation of polyubiquitinated proteins,cell morphological changes, and apoptosis have been reported upontreatment of cells with various proteasome inhibitors. Yet, it should benoted that commercially available proteasome inhibitors inhibit both theconstitutive and immuno-forms of the proteasome. Even bortezomib, theonly FDA-approved proteasome inhibitor for the treatment of relapsedmultiple myeloma patients, does not distinguish between the two forms(Altun et al, Cancer Res 65:7896, 2005). Thus, what is known abouttherapeutic proteasome inhibition is based on work with molecules thatinhibit both forms of the proteasome. Accordingly, compounds of theinvention may be beneficial for reducing the severity of side effectsassociated with molecules that inhibit both forms of the proteasome.

Immunoproteasome expression occurs predominantly in cells and organsthat make up the lymphatic system, such as white blood cells(leukocytes), bone marrow, and the thymus, spleen and lymph nodes.Although some organs preferentially express constitutive proteasomes(e.g., heart), others such as adrenal, liver, lung and gut, appear toexpress both forms.

The immune system, of which leukocytes and lymphoid tissues play a majorrole, is responsible for protecting an organism from outside biologicalinfluences. When functioning properly, it protects the body againstbacterial and viral infections. The immune system also screens forautologous cells that have undergone oncogenic transformation.Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theproteasome is the main provider of these precursor peptides, however,differences between antigenic peptides have been observed between cellswith varying amounts of each proteasome form (Cascio et al, EMBO J20:2357-2366, 2001). In certain embodiments, the invention relates to amethod for inhibiting antigen presentation in a cell, including exposingthe cell to a compound described herein. In certain embodiments, theinvention relates to a method for altering the repertoire of antigenicpeptides produced by the proteasome or other Ntn with multicatalyticactivity. For example, if the activity of the immunoproteasomeproteasome is selectively inhibited, a different set of antigenicpeptides may be produced by the remaining constitutive proteasome andpresented in MHC molecules on the surfaces of cells than would beproduced and presented without any enzyme inhibition.

Several disorders and disease states have been associated with aberrantimmune system function, herein referred to as immune-related conditions.Perhaps the most common immune-related condition is the allergicdisorders such as allergies, asthma and atopic dermatitis like eczema.These occur when the immune system overreacts to exposure to antigens inthe environment. Thus, a further embodiment is a method for suppressingthe immune system of a subject including administering to the subject aneffective amount of a proteasome inhibitor compound in a mannerdescribed herein.

Immunodeficiency disorders occur when a part of the immune system is notworking properly or is not present. They can affect B lymphocytes, Tlymphocytes, or phagocytes and be either inherited (e.g., IgAdeficiency, severe combined immunodeficiency (SCID), thymic dysplasiaand chronic granulomatous) or acquired (e.g., acquired immunodeficiencysyndrome (AIDS), human immunodeficiency virus (HIV) and drug-inducedimmunodeficiencies). A dosing strategy utilizing selective proteasomeinhibitors of the invention may be used to treat immune-relatedconditions such as immunodeficiency disorders.

In autoimmune disorders, the immune system inappropriately attacks thebody's healthy organs and tissues as if they were foreign invaders. Anexample of an autoimmune disease is Sjogren's Syndrome, which ischaracterized by infiltration and focal accumulation of lymphocytes inthe exocrine glands. A study examining the proteasome expression levelrevealed a significant up-regulation of beta5i (LMP7) exclusively in thesalivary glands of SS patients (Egerer et al, Arthritis Rheum 54:1501-8,2006). Other examples of such immune-related conditions include lupus,rheumatoid arthritis, scleroderma, ankylosing spondylitis,dermatomyositis, psoriasis, multiple sclerosis and inflammatory boweldisease (such as ulcerative colitis and Crohn's disease). Tissue/organtransplant rejection occurs when the immune system mistakenly attacksthe cells being introduced to the host's body. Graft versus host disease(GVHD), resulting from allogenic transplantation, arises when the Tcells from the donor tissue go on the offensive and attack the host'stissues. In all three circumstances, autoimmune disease, transplantrejection and GVHD, modulating the immune system by treating the subjectwith a composition of the invention could be beneficial.

Inflammation is the first response of the immune system to infection orirritation. A cellular component of inflammation involves the movementof leukocytes, which express immunoproteasome, from blood vessels intothe inflamed tissue. These cells take on the important role of removingthe irritant, bacteria, parasite or cell debris. Proteasome inhibitorsare already known to have anti-inflammatory activity (Meng et al, PNAS96:10403-10408, 1999). In cases of chronic inflammation, which ischaracterized by a dominating presence of macrophages, the cells thatoriginally served as defensive agents begin to release toxins andcytokines, including TNF-α, now become injurious to the body, resultingin tissue damage and loss. In certain embodiments, the invention relatesto a method of treating inflammation and inflammatory diseasescomprising administering to the subject in need of such treatment aneffective amount of a proteasome inhibitor compound described herein.Inflammatory diseases include acute (e.g., bronchitis, conjunctivitis,pancreatitis) and chronic conditions (e.g., chronic cholecstitis,bronchiectasis, aortic valve stenosis, restenosis, psoriasis andarthritis), along with conditions associated with inflammation such asfibrosis, infection and ischemia.

Following tissue damage, including damage due to the inflammationprocess, progression of regeneration and repair begins. During theregeneration step, lost tissue is replaced by proliferation of cells ofthe same type, which reconstruct the normal architecture. However,improper regeneration of the tissue architecture may have severeconsequences. In some cases of chronic inflammatory liver disease, theregenerated tissue forms an abnormal nodular architecture leading tocirrhosis and portal hypertension. The repair process is where losttissue is replaced by a fibrous scar which is produced from granulationtissue. Fibrosis is the excessive and persistent formation of scartissue resulting from the hyperproliferative growth of fibroblasts andis associated with activation of the TGF-β signaling pathway. Fibrosisinvolves extensive deposition of extracellular matrix and can occurwithin virtually any tissue or across several different tissues.Normally, the level of intracellular signaling protein (Smad) thatactivate transcription of target genes upon TGF-β stimulation isregulated by proteasome activity (Xu et al., 2000). However, accelerateddegradation of the TGF-β signaling components has been observed incancers and other hyperproliferative conditions. Thus, certainembodiments of the invention relate to a method for treatinghyperproliferative conditions such as diabetic retinopathy, maculardegeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy,cirrhosis, biliary atresia, congestive heart failure, scleroderma,radiation-induced fibrosis, and lung fibrosis (idiopathic pulmonaryfibrosis, collagen vascular disease, sarcoidosis, interstitial lungdiseases and extrinsic lung disorders). The treatment of burn victims isoften hampered by fibrosis, thus, in certain embodiments, the inventionrelates to the topical or systemic administration of inhibitors to treatburns. Wound closure following surgery is often associated withdisfiguring scars, which may be prevented by inhibition of fibrosis.Thus, in certain embodiments, the invention relates to a method for theprevention or reduction of scarring.

Infection by bacteria, parasite or virus all result in initiating theinflammatory process. When the resulting inflammation overwhelms thewhole organism, systemic inflammatory response syndrome (SIRS) occurs.The term sepsis is applied when this is due to infection. Overproductionof lipopolysaccharide (LPS)-induced cytokines such as TNFα is consideredto be central to the processes associated with septic shock. Notsurprisingly, LPS also induces an increase in all components of theMHC-1 pathway including the immunoproteasome subunits LMP2 and LMP7(MacAry et al, PNAS 98:3982-3987, 2001). Furthermore, it is generallyaccepted that the first step in the activation of cells by LPS is thebinding of LPS to specific membrane receptors. The α- and β-subunits ofthe 20S proteasome complex have been identified as LPS-binding proteins,suggesting that the LPS-induced signal transduction may be an importanttherapeutic target in the treatment or prevention of sepsis (Qureshi, N.et al., J. Immun. (2003) 171: 1515-1525). Therefore, in certainembodiments, the proteasome inhibitors disclosed herein may be used forthe inhibition of TNFα to prevent and/or treat septic shock.

In another embodiment, the disclosed compositions are useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, the proteasomeinhibitor compositions herein are useful for the treatment of parasiticinfections in humans caused by a protozoan parasite selected fromPlasmodium sps. (including P. falciparum, P. vivax, P. malariae, and P.ovale, which cause malaria), Trypanosoma sps. (including T. cruzi, whichcauses Chagas' disease, and T. brucei which causes African sleepingsickness), Leishmania sps. (including L. amazonesis, L. donovani, L.infantum, L. mexicana, etc.), Pneumocystis carinii (a protozoan known tocause pneumonia in AIDS and other immunosuppressed patients), Toxoplasmagondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the disclosed compositions are useful for thetreatment of parasitic infections in animals and livestock caused by aprotozoan parasite selected from Plasmodium hermani, Cryptosporidiumsps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, andNeurospora crassa. Other compounds useful as proteasome inhibitors inthe treatment of parasitic diseases are described in WO 98/10779, whichis incorporated herein in its entirety.

In certain embodiments, the proteasome inhibitor compositions inhibitproteasome activity in a parasite without recovery in white blood cells.In certain such embodiments, the long half-life of blood cells mayprovide prolonged protection with regard to therapy against recurringexposures to parasites. In certain embodiments, the proteasomeinhibitors described herein may provide prolonged protection with regardto chemoprophylaxis against future infection.

Viral infections contribute to the pathology of many diseases. Heartconditions such as ongoing myocarditis and dilated cardiomyopathy havebeen linked to the coxsackievirus B3. In a comparative whole-genomemicroarray analyses of infected mouse hearts, all three immunoproteasomesubunits were uniformly up-regulated in hearts of mice which developedchronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Someviruses utilize the ubiquitin-proteasome system in the viral entry stepwhere the virus is released from the endosome into the cytosol. Themouse hepatitis virus (MHV) belongs to the Coronaviridae family, whichalso includes the severe acute respiratory syndrome (SARS) coronvirus.Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment ofcells infected with MHV with a proteasome inhibitor resulted in adecrease in viral replication, correlating with reduced viral titer ascompared to that of untreated cells. The human hepatitis B virus (HBV),a member of the Hepadnaviridae virus family, requires virally encodedenvelop proteins to propagate. Inhibiting the proteasome degradationpathway causes a significant reduction in the amount of secretedenvelope proteins (Simsek et al, J Virol 79:12914-12920, 2005). Inaddition to HBV, other hepatitis viruses (A, C, D and E) may alsoutilize the ubiquitin-proteasome degradation pathway for secretion,morphogenesis and pathogenesis.

The bacterium Listeria monocytogenes causes a condition known aslisteriosis, the manifestations of which range from mild (nausea,vomiting and diarrhea) to severe (septicemia, meningitis, encephalitis).A quantitative analysis of changes in proteasome subunit compositionrevealed that infection of mice with lymphocytic choriomeningitis virusor Listeria monocytogenes lead to an almost complete replacement ofconstitutive proteasomes by immunoproteasomes in the liver within sevendays (Khan et al, J Immunol 167:6859-6868, 2001). Prokaryotes have whatis equivalent to the eukaryote 20S proteasome particle. While thesubunit composition of the prokaryote 20S particle is simpler than thatof eukaryotes, it does have the ability to hydrolyze peptide bonds in asimilar manner. For example, the nucleophilic attack on the peptide bondoccurs through the threonine residue on the N-terminus of theβ-subunits. Thus, an embodiment of this invention relates to a method oftreating prokaryotic infections, comprising administering to a subjectan effective amount of a proteasome inhibitor composition disclosedherein. Prokaryotic infections may include diseases caused by eithermycobacteria (such as tuberculosis, leprosy or Buruli Ulcer) orarchaebacteria.

Accordingly, in certain embodiments, the invention relates to a methodfor treating infection (e.g., bacterial, parasitic or viral), includingcontacting a cell with (or administering to a subject) an effectiveamount of a compound disclosed herein.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-Bα, thereby resulting inthe activation of NF-κB (Koong et al., 1994). Interestingly, factorswhich have been identified as being able to enhance immunoproteasomeexpression, TNF-α and lipopolysaccharide, also stimulate NF-κBactivation. It has been demonstrated that the severity of injuryresulting in hypoxia can be reduced with the administration of aproteasome inhibitor (Gao et al., 2000; Bao et al., 2001; Pye et al.,2003). Therefore, certain embodiments of the invention relate to amethod of treating an ischemic condition or reperfusion injurycomprising administering to a subject in need of such treatment aneffective amount of a proteasome inhibitor compound disclosed herein.Examples of such conditions or injuries include, but are not limited to,acute coronary syndrome (vulnerable plaques), arterial occlusive disease(cardiac, cerebral, peripheral arterial and vascular occlusions),atherosclerosis (coronary sclerosis, coronary artery disease),infarctions, heart failure, pancreatitis, myocardial hypertrophy,stenosis, and restenosis.

Cachexia is a syndrome characterized by wasting of skeletal muscleassociated with enhanced proteolysis due to the ubiquitin-proteasomepathway. Inhibiting the proteasome reduces proteolysis, thereby reducingboth muscle protein loss and the nitrogenous load on kidneys or liver(Tawa et al., JCI 100:197-203, 1997). In cachexia, elevated expressionof proinflammatory cytokines, TNF-α and IFN-γ, both of which stimulateexpression of immunoproteasome subunits, have been reported (Acharyya etal., JCI 114:370-378, 2004). In fact, most types of muscle atrophyexhibit elevated rates of protein degradation (Lecker et al., FASEB J18:39-51, 2004). Muscle wasting manifests itself in several lifethreatening diseases, including cancer, sepsis, renal failure, AIDS,fasting, denervation atrophy, acidosis, diabetes, disuse atrophy andcongestive heart failure. One embodiment of the invention relates to thetreatment of cachexia and muscle-wasting diseases. Methods of theinvention are useful for treating conditions such as cancer, chronicinfectious diseases, fever, muscle disuse (atrophy) and denervation,nerve injury, fasting, renal failure associated with acidosis, andhepatic failure. See, e.g., Goldberg, U.S. Pat. No. 5,340,736.

Degradation of certain proteins by the proteasome effect signalingmechanisms that, in turn, effect gene transcription, cell cycle andmetabolic pathways. As noted above, proteasome inhibitors block bothdegradation and processing of ubiquitinated NF-κB in vitro and in vivo.Proteasome inhibitors also block IκB-A degradation and NF-κB activation(Palombella, et al. Cell (1994) 78:773-785; and Traenckner, et al., EMBOJ. (1994) 13:5433-5441). One embodiment of the invention is a method forinhibiting IκB-α degradation, including contacting the cell with acompound described herein.

In certain embodiments, the invention relates to methods for affectingcyclin-dependent eukaryotic cell cycles, including exposing a cell (invitro or in vivo) to a proteasome inhibitor disclosed herein. Cyclinsare proteins involved in cell cycle control. The proteasome participatesin the degradation of cyclins. Examples of cyclins include mitoticcyclins, G1 cyclins, and cyclin B. Degradation of cyclins enables a cellto exit one cell cycle stage (e.g., mitosis) and enter another (e.g.,division). It is believed all cyclins are associated with p34^(cdc2)protein kinase or related kinases. The proteolysis targeting signal islocalized to amino acids 42-RAALGNISEN-50 (destruction box). There isevidence that cyclin is converted to a form vulnerable to a ubiquitinligase or that a cyclin-specific ligase is activated during mitosis(Ciechanover, A., Cell, (1994) 79:13-21). Inhibition of the proteasomeinhibits cyclin degradation, and therefore inhibits cell proliferation,for example, in cyclin-related cancers (Kumatori et al., Proc. Natl.Acad. Sci. USA (1990) 87:7071-7075). In certain embodiments, theinvention relates to a method for treating a proliferative disease in asubject (e.g., cancer, psoriasis, or restenosis), comprisingadministering to the subject an effective amount of a proteasomeinhibitor composition in a manner disclosed herein. The invention alsorelates to a method for treating cyclin-related inflammation in asubject, comprising administering to a subject a therapeuticallyeffective amount of a proteasome inhibitor composition in a mannerdescribed herein.

In maturing reticulocytes and growing fibroblasts, cells deprived ofinsulin or serum, the rate of proteolysis nearly doubles, suggesting arole for the proteasome in cellular metabolism. In certain embodiments,the invention relates to methods for reducing the rate of intracellularprotein degradation in a cell. Each of these methods comprisescontacting a cell (in vivo or in vitro, e.g., a muscle in a subject)with an effective amount of a pharmaceutical composition comprising aproteasome inhibitor disclosed herein.

Alzheimer's disease (AD) is a progressive neurodegenerative diseasedisorder associated with a loss of higher cognitive function.Pathological hallmarks of the disease include senile amyloid plaques,neurofibrillary tangles, dystrophic neuritis and significant neuronalloss in selected regions of the brain. Microglia, the residentmacrophages in the brain, release numerous proinflammatory cytokines,including TNF-α, when activated by Aβ42, a peptide associated withneuritic and vascular amyloid plaques. This microglial-mediatedinflammatory response contributes to significant neuronal loss.Cell-based studies demonstrated that primary cortical neurons treatedwith conditioned media from microglial BV2 cells stimulated either withLPS/INF-γ or sonicated Aβ42 peptides resulted in approximately a 60%decrease in cell viability (Gan et al., J. Biol. Chem. 279:5565-5572,2004). A higher expression of immunoproteasome is found in brain tissuefrom AD patients than in that of non-demented elderly adults (Mishto etal, Neurobiol Aging 27:54-66, 2006).

Patients suffering from Huntington's disease (HD), anotherneurodegenerative disorder, display motor dysfunction and cognitivedecline over a period of years until death. Upon autopsy, the presenceof inclusions or intraneuronal aggregates, caused by a polyQ expansionmutation (also referred to as a CAG triplet repeat expansion), can bedetected, accompanied by significant atrophy in the striatum and cortexportions of the brain. Immunohistochemistry revealed that there was asignificant enhancement in immunoproteasome expression in the striatumand frontal cortex of brains from HD patients as compared to those fromage-matched normal adults (Diaz-Hernandez et al, J Neurosci23:11653-1161, 2003). Upon further analysis, it was discovered that theenhancement predominantly occurred in the degenerating neurons. Using amouse model of HD, the researchers noted a selective increase in bothchymotrypsin- and trypsin-like activities in the affected andaggregate-containing regions of the brain, primarily the cortex andstriatum (Diaz-Hernandez et al, J Neurosci 23:11653-1161, 2003).

Accordingly, certain embodiments of the invention relate to the use ofproteasome inhibitor compositions disclosed herein for the treatment ofneurodegenerative diseases. Neurodegenerative diseases and conditionsincludes, but not limited to, stroke, ischemic damage to the nervoussystem, neural trauma (e.g., percussive brain damage, spinal cordinjury, and traumatic damage to the nervous system), multiple sclerosisand other immune-mediated neuropathies (e.g., Guillain-Barre syndromeand its variants, acute motor axonal neuropathy, acute inflammatorydemyelinating polyneuropathy, and Fisher Syndrome), HIV/AIDS dementiacomplex, axonomy, diabetic neuropathy, Parkinson's disease, Huntington'sdisease, multiple sclerosis, bacterial, parasitic, fungal, and viralmeningitis, encephalitis, vascular dementia, multi-infarct dementia,Lewy body dementia, frontal lobe dementia such as Pick's disease,subcortical dementias (such as Huntington or progressive supranuclearpalsy), focal cortical atrophy syndromes (such as primary aphasia),metabolic-toxic dementias (such as chronic hypothyroidism or B12deficiency), and dementias caused by infections (such as syphilis orchronic meningitis).

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed proteasome inhibitor compositions may be useful in thetreatment and/or prevention of diseases associated with bone loss, suchas osteoporosis.

Cancer is a general term for disease characterized by uncontrolled,abnormal growth of cells. Many cancers arise via multistep pathwaysinvolving inactivation of tumor suppressor proteins and activation ofoncogenic peptides. Cancer cells can spread to other parts of the bodythrough the lymphatic system or blood stream. Usually, cancer isclassified according to the type of tissue or cell most prominentlyinvolved. As noted previously, proteasome inhibition has already beenvalidated as a therapeutic strategy for the treatment of cancer,particularly multiple myeloma. As shown in FIG. 1, multiple myelomacells possess both forms of the proteasome, although the ratio can varysomewhat. Multiple myeloma is a hematologic disease characterized by anexcessive number of abnormal plasma cells in the bone marrow. Plasmacells develop from B-cells, thus it is not surprising that other B-cellmalignancies would also express immunoproteasome to some extent. Exceptfor two chronic mylogenous leukemia cell lines, heme-related cancers(e.g., multiple myeloma, leukemias and lymphomas) generally appear toexpress immunoproteasome (FIG. 1). Cancer cells originating fromlymphoid cells express 30% or more immunoproteasome. In certainembodiments, the invention relates to a method for the treatment ofcancer, comprising administering a therapeutically effective amount of acompound described herein. In certain preferred embodiments, the canceris a heme-related disorder.

Intriguingly, some cancers (e.g., solid tumors, head and neck squamouscell carcinoma, cervical carcinoma and small cell lung carcinoma) appearto have down regulated immunoproteasome expression (Evans et al, JImmunol 167:5420, 2001; Meissner et al, Clin Cancer Res 11:2552, 2005;Restifo et al, J Exp Med 177:265-272, 1993). This appears to becorrelated with deficient antigen processing and may be a strategy usedby tumor cells to escape immune surveillance. The treatment of the cellswith INF-γ could induce immunoproteasome expression. Therefore, certainembodiments of the invention relate to a method of treating cancerscomprising administering to a subject in need of such treatment aneffective amount of INF-γ or TNF-α and a proteasome inhibitor compounddisclosed herein.

Administration

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteasomeinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential, or separate dosing of the individualcomponents of the treatment.

In certain embodiments, a compound of the invention is conjointlyadministered with one or more other proteasome inhibitor(s).

In certain embodiments, a compound of the invention is conjointlyadministered with a chemotherapeutic. Suitable chemotherapeutics mayinclude, natural products such as vinca alkaloids (i.e. vinblastine,vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e.etoposide, teniposide), antibiotics (dactinomycin (actinomycin D)daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone,bleomycins, plicamycin (mithramycin) and mitomycin, enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates(busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin),trazenes-dacarbazinine (DTIC); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate), pyrimidineanalogs (fluorouracil, floxuridine, and cytarabine), purine analogs andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane,and letrozole); and platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones (i.e. estrogen) and hormone agonists such as leutinizinghormone releasing hormone (LHRH) agonists (goserelin, leuprolide andtriptorelin). Other chemotherapeutic agents may include mechlorethamine,camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine,or any analog or derivative variant of the foregoing.

In certain embodiments, a compound of the invention is conjointlyadministered with a steroid. Suitable steroids may include, but are notlimited to, 21-acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, meprednisone, methylprednisolone,mometasone furoate, paramethasone, prednicarbate, prednisolone,prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate,prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide,triamcinolone hexacetonide, and salts and/or derivatives thereof.

In certain embodiments, a compound of the invention is conjointlyadministered with an immunotherapeutic agent. Suitable immunotherapeuticagents may include, but are not limited to, cyclosporine, thalidomide,and monoclonal antibodies. The monoclonal antibodies can be either nakedor conjugated such as rituximab, tositumomab, alemtuzumab, epratuzumab,ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab,erlotinib and trastuzumab.

EXEMPLIFICATION

Synthesis of (A)

To a solution of Cbz-Trp-OH (10 g, 29 mmol) in DMF (150 mL) was addedMe-Im (25.2 mL, 458 mmol) dropwise. The solution was allowed to stir for10 minutes followed by the addition of TBSCl (23.9 g, 158 mmol). Theresulting solution was allowed to stir overnight. Water (100 mL) wasthen added and the mixture was extracted with EtOAc (3×50 mL). Thecombined organic layers were washed with water (3×50 mL) and brine (50ml), dried over MgSO₄, filtered, and concentrated under reduced pressureto yield an oil that was dissolved in 1:1 water/THF (200 mL) and K₂CO₃(440 mg, 0.031 mmol) was added to the solution. The resulting solutionwas allowed to stir overnight. The organic solvent was then removedunder reduced pressure and the pH adjusted to 2 with 1N HCl. Theresulting solution was extracted with EtOAc (3×75 mL) and the combinedorganic layers were dried over MgSO₄, filtered, and concentrated underreduced pressure to yield (A).

Synthesis of (B)

To a solution of dimethyl hydroxylamine hydrochloride (3.6 g, 36.9 mmol)in DCM (20 mL) at 0° C. was added diisopropylethylamine (DIEA) (5 mL,54.7 mmol) dropwise. The resulting solution was allowed to stir for 20minutes.

To a solution of (A) in DCM (20 mL) at 0° C. was addedisobutylchlorformate (IBCF) (5 mL, 51.5 mmol) dropwise, followed by thedrop wise addition of N-methylmorpholine (NMM) (4.0 mL, 56.7 mmol). Theresulting solution was allowed to stir for 10 minutes before adding itto the previously prepared dimethyl hydroxylamine hydrochloride/DIEAsolution. The mixture was allowed to stir for 3 hrs at 0° C., followedby the addition of water (50 mL). The layers were separated and thewater layer was washed with DCM (3×15 mL). The combined organic layerswere washed with 1N HCl (3×20 mL) and brine (20 mL), dried over MgSO₄,filtered and concentrated under reduced pressure to yield an oil thatwas purified by flash chromatography using 20 to 40% EtOAc/hexanes asthe eluent to yield (B).

Synthesis of (C)

To a solution of (B) (6.82 g, 14.4 mmol) in a solution of THF (40 mL) at−20° C. was added a solution isopropenyl magnesium bromide (90 mL, 0.5 Min THF) while keeping the internal temperature below −5° C. The solutionwas allowed to stir for 3 hrs at 0° C. followed by the addition of 1NHCl (20 mL). The solution was filtered through Celite 521 and the filtercake washed with EtOAc. The organic solvent was then removed underreduced pressure and the remaining aqueous solution was extracted withEtOAc (3×20 mL). The combined organic layers were washed with sat.NaHCO₃ (3×, 15 mL) and brine (15 mL), dried over MgSO₄, filtered, andconcentrated under reduced pressure to provide an oil that was purifiedby flash chromatography using 10 to 20% EtOAc/hexanes as the eluent toyield (C).

Synthesis of (D) and (E)

To a solution of (C) (3.06 g, 6.75 mmol) in MeOH (40 mL) and THF (40 mL)was added CeCl₃.7H₂O (3.64 g, 9.77 mmol). The resulting mixture wasallowed to stir until it became homogenous. The solution was then cooledto 0° C. and NaBH₄ (369 mg, 9.75 mmol) was added over 10 minutes. Thesolution was allowed to stir for 1 hr followed by the addition of AcOH(5 mL) with continued stirring for 20 minutes. The solvent wasevaporated under reduced pressure and the resulting residue was dilutedwith water (30 mL) and extracted with EtOAc (3×10 mL). The combinedorganic layers were washed with water (3×10 mL) and brine (10 mL), driedover MgSO₄, filtered, and concentrated under reduced pressure to yield a4/1 mixture of (D) and (E).

Synthesis of (F) and (G)

To a solution of (D) and (E) in DCM (90 mL) at 0° C. was added VO(acac)₂(63 mg, 0.23 mmol), after stirring for 5 minutes t-BuOOH (2.25 mL, 6.0Min decane) was added dropwise. The resulting solution was allowed tostir for 2 hrs and was then filtered through Celite 521, and the filtercake was washed with DCM (20 mL). The combined organic layers werewashed with sat. NaHCO₃ (3×20 mL) and brine (20 mL), dried over MgSO₄,filtered, and concentrated under reduced pressure to yield a 4/1 mixtureof (F) and (G).

Synthesis of (H)

To a solution of Dess-Martin periodinane (6.75 g, 15.9 mmol) in DCM (75mL) at 0° C. was added a solution of (F) and (G) in DCM (35 mL)dropwise. The solution was allowed to warm to room temperature and stirovernight. The solvent was then concentrated under reduced pressure andthe residue was diluted with EtOAc (20 mL) and sat. NaHCO₃ ₍20 mL). Theresulting mixture was filtered through Celite 521 and the filter cakewas washed with EtOAc (20 mL). The layers were separated and the organiclayer was washed with water (3×10 mL) and brine (10 mL), dried overMgSO₄, filtered, and concentrated under reduced pressure to provide anmixture of (H) and (I) (4/1) that was purified by flash chromatographyusing 15 to 40% EtOAc/hexanes as the eluent to yield (H).

Synthesis of 1

To a solution of (H) (50 mg, 1.06 mmol) in TFA (5 mL) was added Pd/C (14mg, 10%). The resulting mixture was allowed to stir under 1 atmosphereof H₂ for 2 hrs followed by dilution with DCM (10 mL). The mixture wasfiltered through Celite 521 and the filter cake was washed with DCM (10mL). The filtrate was concentrated under reduced pressure and theresulting residue was diluted with DCM (10 mL) and concentrated underreduced pressure a second time. The residue was placed under high vacuumfor 2 hrs to provide 1.

Synthesis of (J)

To a solution of Fmoc-Trp (Boc)-OH (2.4 mmol, 1.0 g,) in DCM (20 mL) wasadded Me-Im (6.7 mmol, 0.370 mL) and the mixture was stirred until thesolution was homogenous, at which time1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) (2.9 mmol, 0.870g,) was added. Once the MSNT had dissolved, the reaction mixture wasadded to HMPB-BHA resin (0.8 mmol, 1.25 g) and the resulting solutionwas allowed to shake for 45 minutes. The resin was filtered and washedwith DMF (50 mL), MeOH (50 mL), and DCM (50 mL). The resin was thenallowed to air dry to provide (J).

Synthesis of (K)

To (J) (0.40 mmol, 0.62 g) was added 20% piperidine/DMF (10 mL) and theresulting heterogeneous solution was allowed to shake for 20 minutes.The mixture was filtered and the resin was washed with DMF (20 mL), MeOH(20 mL), and DCM (20 mL) and allowed to air dry before subjecting it tothe above reaction condition a second time to yield (K).

Synthesis of (L)

To (K) (0.40 mmol) was added DMF (20 mL), Cbz-D-Ala-OH (0.40 mmol, 0.090g), DIEA (1.6 mmol, 0.12 mL), HOBT (0.64 mmol, 0.062 mg), and BOP (0.64mmol, 0.178 g) and the reaction mixture was allowed to shake for 45minutes. The reaction mixture was filtered and the resin was washed withDMF (40 mL), MeOH (40 mL), and DCM (40 mL), and allowed to air dry, toyield (L).

Synthesis of (M)

To (L) (0.08 mmol) was added 5% TFA/DCM (2 mL) and the mixture wasallowed to shake for 20 minutes. The reaction was filtered and the resinwas washed with DCM (10 mL). The volatiles were removed under reducedpressure and the resulting oil was diluted with DCM (10 mL) andevaporated a total of three times to yield (M).

Synthesis of (N)

To a stirred solution of (M) (0.11 mmol, 0.019 g) in MeCN (4 mL) and DMF(1 mL) was added (M) (0.1 mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2mmol, 0.032 g), and HBTU (0.23 mmol, 0.087 g) and the mixture wasstirred at room temperature overnight. The reaction was diluted with satNaHCO₃ (15 mL) and extracted with EtOAc. The organic layer was washedwith sat. NaHCO₃ and brine, dried over MgSO₄, and the volatiles removedunder reduced pressure. The crude material was purified by flashchromatography using 20 to 40% EtOAc/hexanes as the eluent to afford(N).

Synthesis of 2

To a stirred solution of (N) (0.1 mmol) in pyridine (1.5 mL) and THF(3.0 mL) at 0° C. was added a solution of HF/pyridine dropwise. Thesolution was allowed to stir for 2 hours at 0° C. prior to the additionof water (5.0 mL) and extraction with EtOAc. The combined organic layerswere washed with sat. NaHCO₃ and brine, dried over MgSO₄, filtered andthe volatiles removed under reduced pressure. The crude material waspurified by flash chromatography using 30 to 60% EtOAc/hexanes as theeluent, to afford 2 (4.2 mg).

Synthesis of (O)

To a solution of Fmoc-Tyr(Me)-OH (1.9 mmol, 0.80 g,) in DCM (20 mL) wasadded Me-Im (6.7 mmol, 0.370 mL). When the solution was homogenous, MSNT(2.9 mmol, 0.870 g,) was added and the mixture was stirred until theMSNT dissolved, at which time HMPB-BHA resin (0.64 mmol, 1.00 g) and theresulting solution was allowed to shake for 45 minutes. The resin wasfiltered and washed with DMF (50 mL), MeOH (50 mL), and DCM (50 mL) andthen the resin was allowed to air dry, to yield (O).

Synthesis of (P)

To (O) (0.40 mmol, 0.62 g) was added 20% piperidine/DMF (10 mL) and theresulting heterogenous solution was allowed to shake for 20 minutes. Themixture was filtered and the resin was washed with DMF (20 mL), MeOH (20mL), and DCM (20 mL) and allowed to air dry. The resin was thensubjected to the above reaction condition a second time to yield (P).

Synthesis of (Q)

To (P) (0.40 mmol) was added DMF (20 mL), Cbz-D-Ala-OH (0.40 mmol, 0.090g), DIEA (1.6 mmol, 0.12 mL), HOBT (0.64 mmol, 0.062 mg), and BOP (0.64mmol, 0.178 g) and the reaction mixture was allowed to shake for 45minutes. The reaction mixture was filtered and the resin was washed withDMF (40 mL), MeOH (40 mL), and DCM (40 mL), and allowed to air dry, toyield (Q).

Synthesis of (R)

To (Q) (0.08 mmol) was added 5% TFA/DCM (2 mL) and the mixture wasallowed to shake for 20 minutes. The reaction was filtered and the resinwas washed with DCM (10 mL). The volatiles were then removed underreduced pressure and the resulting oil was diluted with DCM (10 mL) andevaporated a total of three times to yield (R).

Synthesis of (S)

To a stirred solution of 1 (0.11 mmol, 0.019 g) in MeCN (4 mL) and DMF(1 mL) was added (R) (0.1 mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2mmol, 0.032 g), and HBTU (0.23 mmol, 0.087 g) and the mixture wasstirred at room temperature overnight. The reaction was diluted with satNaHCO₃ (15 mL) and extracted with EtOAc. The organic layer was washedwith sat. NaHCO₃, and brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The resulting material was purifiedby flash chromatography using 20 to 40% EtOAc/hexanes as the eluent toafford (S).

Synthesis of 3

To a stirred solution of (S) (0.1 mmol), in pyridine (1.5 mL) and THF(3.0 mL) at 0° C. was added a solution of HF/Pyridine dropwise. Thesolution was allowed to stir for 2 hours at 0° C. followed by theaddition of water (5.0 mL) and extraction with EtOAc. The organic layerwas then washed with sat. NaHCO₃, and brine, dried over MgSO₄, filtered,and concentrated under reduced pressure. The resulting material waspurified by flash chromatography using 30 to 60% EtOAc/hexanes as theeluent, to afford 3 (6.7 mg).

Synthesis of (U)

To a solution of dimethyl hydroxylamine hydrochloride (18.4 g, 226.3mmol) in DCM (400 mL) at 0° C. was added DIEA (25.8 mL, 282 mmol)dropwise. The resulting solution was allowed to stir for 20 minutes.

To a solution of Cbz-Phe-OH (50 g, 169 mmol) in DCM (400 mL) at 0° C.was added IBCF (24.4 mL, 266 mmol) dropwise, followed by the dropwiseaddition of NMM (20.7 mL, 293 mmol). The resulting solution was allowedto stir for 10 minutes then added to the previously prepared dimethylhydroxylamine hydrochloride/DIEA solution. The mixture was allowed tostir for 3 hrs at 0° C. followed by the addition of water (250 mL). Thelayers were then separated and the water layer washed with DCM (3×100mL). The combined organic layers were washed with 1N HCl (3×100 mL) andbrine (100 mL), dried over MgSO₄, filtered, and concentrated underreduced pressure to yield an oil that was purified by flashchromatography using 20 to 40% EtOAc/hexanes as the eluent, to yield(U).

Synthesis of (V)

To a solution of (U) (47 g, 145 mmol) in a solution of THF (400 mL) at−20° C. was added a solution isopropenyl magnesium bromide (800 mL, 0.5M in THF) while keeping the internal temperature below −5° C. Thesolution was allowed to stir for 3 hrs at 0° C. followed by the additionof 1N HCl (200 mL). The solution was filtered through Celite 521 and thefilter cake washed with EtOAc. The organic solvent was then removedunder reduced pressure and the remaining aqueous solution was extractedwith EtOAc (3×200 mL). The combined organic layers were washed withsatd. NaHCO₃ (3×150 mL) and brine (150 mL), dried over MgSO₄, filtered,and concentrated under reduced pressure to yield an oil that waspurified by flash chromatography using 20 to 40% EtOAc/hexanes as theeluent to yield (V).

Synthesis of (W) and (X)

To a solution of (V) (30.03 g, 92.0 mmol) in MeOH (500 mL) and THF (500mL) was added CeCl₃.7H₂O (48.04 g, 130 mmol). The resulting solution wasallowed to stir until it became homogenous. The solution was then cooledto 0° C. and NaBH₄ (4.90 mg, 129 mmol) was added over a 10 minuteperiod. The solution was allowed to stir for 1 hr followed by theaddition of AcOH (70 mL) with continued stirring for 20 minutes. Themixture was then concentrated under reduced pressure and the resultingresidue diluted with water (400 mL) and extracted with EtOAc (3×130 mL).The combined organic layers were washed with water (3×, 130 mL) andbrine (130 mL), dried over MgSO₄, filtered, and concentrated underreduced pressure to yield a 5/1 mixture of (W) and (X).

Synthesis of (Y) and (Z)

To a solution of (W) and (X) in DCM (500 mL) at 0° C. was addedVO(acac)₂ (900 mg, 3.26 mmol), after stirring for 5 minutes t-BuOOH (30mL, 6.0M in decane) was added dropwise. The resulting solution wasallowed to stir for 2 hrs then filtered through Celite 521 and thefilter cake was washed with DCM (200 mL). The filtrate was then washedwith satd. NaHCO₃ (3×200 mL) and brine (200 mL), dried over MgSO₄,filtered, and concentrated under reduced pressure to yield a 5/1 mixtureof (Y) and (Z).

Synthesis of (AA)

To a solution of Dess-Martin periodinane (40 g, 94.2 mmol) in DCM (300mL) at 0° C. was added a solution of (Y) and (Z) in DCM (100 mL)dropwise. The solution was then allowed to warm to room temperature andstir overnight. The reaction mixture was then concentrated under reducedpressure and the residue diluted with EtOAc (120 mL) and satd. NaHCO₃(120 mL). The resulting mixture was filtered through Celite 521 and thefilter cake washed with EtOAc (120 mL). The layers were separated andthe organic layer was washed with water (3×60 mL) and brine (60 mL),dried over MgSO₄ filtered, and concentrated under reduced pressure togive an oil that was purified by flash chromatography using 15 to 40%EtOAc/hexanes as the eluent to yield (AA).

Synthesis of 4

To a solution of (AA) (50 mg, 1.06 mmol) in TFA (5 mL) was added Pd/C(14 mg, 10%). The resulting mixture was allowed to stir under 1atmosphere H₂ for 2 hrs, and then diluted with DCM (10 mL). The mixturewas filtered through Celite 521 and the filter cake washed with DCM (10mL). The filtrate was then concentrated under reduced pressure and theresidue diluted with DCM (10 mL) and concentrated under reduced pressurea second time. The residue was placed under high vacuum for 2 hrs toyield 4.

Synthesis of (CC)

To (BB) (0.06 mmol) was added DMF (2 mL), Cbz-D-Abu-OH (0.12 mmol, 0.032g), DIEA (0.256 mmol, 0.075 mL), HOBT (0.102 mmol, 0.010 mg), and BOP(0.102 mmol, 0.075 g) and the reaction mixture was allowed to shake for45 minutes. The reaction mixture was then filtered and the resin washedwith DMF (4 mL), MeOH (4 mL), and DCM (4 mL), and allowed to air dry, toyield (CC).

Synthesis of (DD)

To (CC) (0.08 mmol) was added 50% TFA/DCM (2 mL) and the mixture wasallowed to shake for 20 minutes. The reaction was filtered and the resinwas washed with DCM (10 mL). The solution was then under reducedpressure and the resulting oil was diluted with DCM (10 mL) andevaporated a total of three times to yield (DD).

Synthesis of 5

To a stirred solution of 4 (0.11 mmol, 0.019 g) in MeCN (4 mL) and DMF(1 mL) was added (DD) (0.1 mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2mmol, 0.032 g), and HBTU (0.23 mmol, 0.087 g) and the mixture wasstirred at room temperature overnight. The reaction was then dilutedwith sat NaHCO₃ (15 mL) and extracted with EtOAc. The organic layer waswashed with satd. NaHCO₃ and brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The resulting material was purifiedby flash chromatography using 25 to 55% EtOAc/hexanes as the eluent toafford 5 (12.0 mg).

Synthesis of (EE)

To (BB) (0.06 mmol) was added DMF (2 mL), Cbz-D-Leu-OH (0.12 mmol, 0.032g), DIEA (0.256 mmol, 0.075 mL), HOBT (0.102 mmol, 0.010 mg), and BOP(0.102 mmol, 0.075 g) and the reaction mixture was allowed to shake for45 minutes. The reaction mixture was then filtered and the resin washedwith DMF (4 mL), MeOH (4 mL), and DCM (4 mL), and allowed to air dry, toyield (EE).

Synthesis of (FF)

To (FF) (0.08 mmol) was added 50% TFA/DCM (2 mL) and the mixture wasallowed to shake for 20 minutes. The reaction was filtered and the resinwashed with DCM (10 mL). The volatiles were removed under reducedpressure and the resulting oil was diluted with DCM (10 mL) andevaporated a total of three times to yield (FF).

Synthesis of 6

To a stirred solution of 4 (0.11 mmol, 0.019 g) in MeCN (4 mL) and DMF(1 mL) was added (FF) (0.1 mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2mmol, 0.032 g), and HBTU (0.23 mmol, 0.087 g) and the mixture wasstirred at room temperature overnight. The reaction was then dilutedwith sat NaHCO₃ (15 mL) and extracted with EtOAc. The organic layer waswashed with satd. NaHCO₃ and brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The resulting material was thenpurified by flash chromatography using 25 to 55% EtOAc/hexanes as theeluent to afford 20 (14.0 mg).

Synthesis of 7

To a stirred solution of 4 (0.11 mmol, 0.019 g) in MeCN (4 mL) and DMF(1 mL) was added (R) (0.1 mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2mmol, 0.032 g), and HBTU (0.23 mmol, 0.087 g) and the mixture wasstirred at room temperature overnight. The reaction was then dilutedwith satd. NaHCO₃ (15 mL) and extracted with EtOAc. The organic layerwas washed with satd. NaHCO₃ and brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The resulting material was purifiedby flash chromatography using 25 to 55% EtOAc/hexanes as the eluent toafford 7 (10.5 mg).

Synthesis of (HH)

To a solution of dimethyl hydroxylamine hydrochloride (331 mg, 3.4 mmol)in DCM (20 mL) at 0° C. was added triethylamine (343 mg, 3.4 mmol)dropwise. The resulting solution was allowed to stir for 20 minutes.

To a solution of Cbz-HomoPhe-OH (1.0 g, 3.2 mmol) in DCM (100 mL) at 0°C. was added IBCF (460 mg, 3.35 mmol) dropwise, followed by the dropwiseaddition of NMM (343 mg, 3.4 mmol). The resulting solution was allowedto stir for 10 minutes and then added to the previously prepareddimethyl hydroxylamine HCl/TEA solution. The resulting mixture wasstirred at 0° C. for 3 hrs followed by the addition of water (50 mL).The layers were separated and the aqueous layer was extracted with DCM(3×100 mL). The combined organic layers were washed with 1N HCl (30 mL)and brine (30 mL), dried over MgSO₄, filtered, and concentrated underreduced pressure. The resulting material was then purified by flashchromatography using EtOAc/hexanes (1:3) as the eluent to yieldintermediate (HH) (0.92 g).

Synthesis of (II)

To a solution of (HH) (920 mg, 2.6 mmol) in THF (50 mL) at −20° C. wasadded a solution of isopropenyl magnesium bromide (26 mL, 12.9 mmol, 0.5M in THF). The resulting solution was allowed to stir at 0° C. for 6hours followed by the addition of 1N HCl (10 mL). The resulting mixturewas filtered through Celite 521 and the filter cake was washed withethyl acetate. The layers were separated and aqueous phase was extractedwith ethyl acetate (3×20 mL). The combined organic layers were washedwith satd. NaHCO₃ (30 mL) and brine (30 mL), dried over MgSO₄, filteredand concentrated under reduced pressure. The resulting material waspurified by flash chromatography using EtOAc/hexanes (1:3) as the eluentto yield (II) (700 mg).

Synthesis of (JJ) and (KK)

To a solution of (II) (700 mg, 2.1 mmol) in DCM (50 mL) at 0° C. wasadded CeCl₃.7H₂O (942 mg, 2.52 mmol) and NaBH₄ (98 mg, 2.52 mmol)successively. The solution was stirred at room temperature overnightfollowed by the addition of AcOH (5 mL). The mixture was concentratedunder reduced pressure and then diluted with EtOAc (100 mL) and satd.NaHCO₃ (50 mL). The aqueous layer was then extracted with EtOAc (2×50mL) and the combined organic layers were dried over Na₂SO₄, filtered,and concentrated under reduced pressure to a yellow oil that waspurification by flash chromatography using EtOAc/hexanes (1:3) as theeluent to yield (JJ) and (KK) in a 5/1 ratio.

Synthesis of (LL) and (MM)

To a solution of (JJ) and (KK) in THF (50 mL) at 0° C. was addedVO(acac)₂ (18 mg, 0.066 mmol) and t-BuOOH (0.9 mL, 6.0M in decane)successively. The resulting solution was stirred at room temperature for10 hours then filtered through Celite 521 and the filter cake was washedwith EtOAc (100 mL). The combined organic layers were washed with satd.NaHCO₃ (10 mL) and brine (10 mL), dried over MgSO₄, filtered andconcentrated under reduced pressure to give (LL) and (MM) (585 mg) in a5/1 ratio.

Synthesis of (NN)

To a solution of Dess-Martin Periodinane (1.40 g, 3.3 mmol) in DMSO (20mL) at 0° C. was added (LL) and (MM) (585 mg) in DMSO (10 mL). Thesolution was stirred at room temperature for 6 hours, and then dilutedwith EtOAc (100 mL) and satd. NaHCO₃ (50 mL), the aqueous phase was thenextracted with EtOAc (2×50 mL), and the combined organic layers weredried over Na₂SO₄, filtered, and concentrated under reduced pressure togive a yellow oil that was purified by flash chromatography usingEtOAc/hexanes (2:3) as the eluent to yield (NN) (465 mg).

Synthesis of 8

To a solution of (NN) (290 mg, 0.82 mmol) in TFA (5 mL) was added Pd/C(14 mg, 10%). The resulting mixture was allowed to stir under 1atmosphere H₂ for 2 hrs, and was then diluted with DCM (10 mL). Themixture was filtered through Celite 521 and the filter cake washed withDCM (10 mL). The was concentrated under reduced pressure and the residuediluted with DCM (10 mL) and concentrated under reduced pressure. Theresulting residue was placed under high vacuum for 2 hrs, to yield 8.

Synthesis of (OO)

To (K) (0.06 mmol) was added DMF (2 mL), Cbz-Ala-OH (0.12 mmol, 0.032g), DIEA (0.256 mmol, 0.075 mL), HOBT (0.102 mmol, 0.010 mg), and BOP(0.102 mmol, 0.075 g) and the reaction mixture was allowed to shake for45 minutes. The reaction mixture was then filtered and the resin washedwith DMF (4 mL), MeOH (4 mL), and DCM (4 mL), and allowed to air dry, toyield (OO).

Synthesis of (PP)

To (OO) (0.08 mmol) was added 50% TFA/DCM (2 mL) and the mixture wasallowed to shake for 20 minutes. The reaction was then filtered and theresin washed with DCM (10 mL). The volatiles were removed under reducedpressure and the resulting oil was diluted with DCM (10 mL) andevaporated a total of three times to yield (PP).

Synthesis of 9

To a stirred solution of 8 (0.11 mmol, 0.019 g) in MeCN (4 mL) and DMF(1 mL) was added (PP) (0.1 mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2mmol, 0.032 g), and HBTU (0.23 mmol, 0.087 g) and the mixture wasstirred at room temperature overnight. The reaction was then dilutedwith satd. NaHCO₃ (15 mL) and extracted with EtOAc. The organic layerwas then washed with satd. NaHCO₃ and brine, dried over MgSO₄, filteredand concentrated under reduced pressure. The resulting material waspurified by flash chromatography using 25 to 55% EtOAc/hexanes as theeluent, to afford 9 (7.8 mg).

Synthesis of (QQ)

To a solution of Cbz-Asp (t-Bu)-OH (0.32 mmol, 108 mg) in DMF (2 mL) at0° C. was added HOBT (0.51 mmol, 78 mg), HBTU (0.51 mmol, 194 mg), andDIEA (1.2 mmol, 0.2 mL). Once the resulting mixture became a homogenoussolution, Phe-HMPB-BHA resin (0.13 mmol, 200 mg) was added and theresulting reaction mixture was allowed to shake at 0˜4° C. overnight.The resin was filtered off and washed with DMF (3×5 mL) and DCM (3×5mL). The resin was allowed to air dry to yield (QQ).

Synthesis of (RR)

To (QQ) (0.13 mmol) was added TFA/DCM (5 mL, 5:95) and the mixture wasallowed to shake at 0˜4° C. for 30 minutes. The reaction was thenfiltered and the resin washed with DCM (3×10 mL). The volatiles wereremoved under reduced pressure at 0° C. to yield (RR).

Synthesis of (SS)

To a 0° C. solution of (RR) (0.13 mmol) and 4 (0.12 mmol) in THF (5 mL)was added HOBT (0.18 mmol, 31 mg), HBTU (0.18 mmol, 76 mg) and DIEA (0.6mmol, 0.1 mL), and the resulting reaction mixture was stirred at 0˜4° C.overnight. The reaction mixture was then diluted with EtOAc (100 mL) andsatd. NaHCO₃, and the aqueous phase was extracted with EtOAc. Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to give a yellow oil that waspurified by HPLC, eluting with an MeCN/aq. NH₄OAc solution, to give(SS).

Synthesis of 10

To a 0° C. solution of (SS) in DCM (5 mL) was added TFA acid (5 mL)dropwise and the resulting solution was stirred for 3 hrs. The reactionmixture was then concentrated under reduced pressure and the resultingresidue was purified by HPLC, eluting with an MeCN/aq. NH₄OAc solution,to give 10.

Synthesis of (TT)

To a 0° C. solution of Z-Ala-OH (0.32 mmol, 71 mg) in DMF (2 mL) wasadded HOBT (0.51 mmol, 78 mg), HBTU (0.51 mmol, 194 mg) anddiisopropylethylamine (1.2 mmol, 0.2 mL). Once the resulting mixturebecame homogenous, Phe(4-MeO)-Wang-resin (0.13 mmol, 200 mg) was addedand the resulting reaction mixture was allowed to shake overnight. Theresin was then filtered off and washed with DMF (3×5 mL) and DCM (3×5mL). The resulting resin was allowed to air dry to yield (TT).

Synthesis of (UU)

To (TT) (0.13 mmol) was added 50% TFA/DCM (5 mL) and the mixture wasallowed to shake for 30 minutes. The reaction was then filtered and theresin washed with DCM (3×10 mL). The volatiles were removed underreduced pressure to yield (UU).

Synthesis of 11

To a 0° C. solution of (UU) (0.13 mmol) and 4 (0.12 mmol) in THF (5 mL)was added HOBT (0.18 mmol, 31 mg), HBTU (0.18 mmol, 76 mg) and DIEA (0.6mmol, 0.1 mL). The resulting reaction mixture was stirred at 0˜4° C.overnight followed by dilution with EtOAc (100 mL) and satd. NaHCO₃. Theaqueous phase was then extracted with EtOAc and the combined organiclayers were dried over Na₂SO₄, filtered, and concentrated under reducedpressure to a yellow oil that was purified by HPLC, eluting with anMeCN/aq. NH₄OAc solution, to give 11.

Synthesis of 12

To a 0° C. solution of (VV) (0.18 mmol, 50 mg) and 4 (0.12 mmol) in THF(5 mL) was added HOBT (0.18 mmol, 31 mg), HBTU (0.18 mmol, 76 mg) andDIEA (0.6 mmol, 0.1 mL). The resulting reaction mixture was stirred at0˜4° C. overnight followed by dilution with EtOAc (100 mL) and satd.NaHCO₃. The aqueous layer was then extracted with EtOAc, and thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to a yellow oil that was purified byHPLC, eluting with an MeCN/aq. NH₄OAc solution, to provide 12.

Synthesis of (WW)

To a solution of Fmoc-O-t-butyl-L-tyrosine (6.4 mmol, 2.94 g) in DCM (22mL) was added 1-methylimidazole (4.8 mmol, 0.380 mL) and the mixture wasstirred until the solution was homogenous, at which time1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) (6.4 mmol, 1.9g) was added. Once the MSNT had dissolved, the reaction mixture wasadded to HMPB-BHA resin (1.28 mmol, 2 g) and the resulting mixture wasallowed to shake for 45 minutes. The resin was filtered and washed withDMF (50 mL), MeOH (50 mL), and DCM (50 mL). The resin was then allowedto air dry to provide (WW).

Synthesis of (XX)

To (WW) (0.40 mmol, 0.62 g) was added 20% piperidine/DMF (50 mL) and theresulting mixture was allowed to shake for 20 minutes. The mixture wasfiltered and the resin was washed with DMF (20 mL), MeOH (20 mL), andDCM (20 mL) and allowed to air dry before subjecting it to the abovereaction condition a second time.

To the resulting resin was added DMF (64 mL), Fmoc-Ala-OH (32 mmol, 1.05g), DIEA (12.8 mmol, 2.2 mL), HOBT (5.12 mmol, 692 mg), and HBTU (5.12mmol, 1.94 g) and the reaction mixture was allowed to shake for 45minutes. The reaction mixture was filtered and the resin was washed withDMF (40 mL), DCM (40 mL), MeOH (40 mL), H₂O (40 mL) MeOH (40 mL), H₂O(40 mL), MeOH (40 mL), and DCM (40 mL), and allowed to air dry, to yield(XX).

Synthesis of (YY)

To (XX) (0.192 mmol, 0.3 g) was added 20% piperidine/DMF (10 mL) and theresulting mixture was allowed to shake for 20 minutes. The mixture wasfiltered and the resin was washed with DMF (20 mL), MeOH (20 mL), andDCM (20 mL) and allowed to air dry before subjecting it to the abovereaction condition a second time.

To the resulting resin was added DMF (12 mL), morpholino acetic acid(0.48 mmol, 70 mg), DIEA (1.92 mmol, 334 μL), HOBT (0.768 mmol, 104 mg),and HBTU (0.768 mmol, 291 mg) and the reaction mixture was allowed toshake for 45 minutes. The reaction mixture was filtered and the resinwas washed with DMF (40 mL), DCM (40 mL), MeOH (40 mL), H₂O (40 mL),MeOH (40 mL) H₂O (40 mL), MeOH (40 mL), and DCM (40 mL), and allowed toair dry, to yield (YY).

Synthesis of (ZZ)

To (YY) (0.192 mmol) was added 5% TFA/DCM (10 mL) and the mixture wasallowed to shake for 10 minutes at 0° C. The reaction mixture wasfiltered and the resin was washed with DCM (10 mL). The volatiles wereremoved under reduced pressure and the resulting oil was diluted withDCM (10 mL) and evaporated a total of three times to yield (ZZ).

Synthesis of 13

To a stirred solution of (ZZ) (0.192 mmol, 83 mg) in MeCN (6 mL) and DMF(2 mL) was added 4 (0.384 mmol, 79 mg), DIEA (0.768 mmol, 133 μL), HOBT(0.3 mmol, 41 mg), and HBTU (0.3 mmol, 116 mg) and the mixture wasstirred at 0° C. for 2 hours. The reaction was diluted with sat NaHCO₃(15 mL) and extracted with EtOAc (3×). The organic layer was washed withsat. NaHCO₃ and brine, dried over MgSO₄, filtered, and concentratedunder reduced pressure. The resulting crude material was purified byflash chromatography using EtOAc then EtOAc/MeOH/TEA (98/1/1) as theeluent to afford 13 as a white solid that was characterized by LC/MS(LCRS (MH) m/z: 623.80).

Synthesis of (AAA)

A suspension of Boc-Tyr(Me)-OH (10 g) in anhydrous dichlormethane (450mL) was cooled to −5° C. in an ice/acetone bath. To this suspension wasadded triethylamine (9.4 mL, 67.8 mmol) and DMAP (600 mg). A solution ofbenzylchloroformate (5.7 mL, 40.6 mmol) in dichloromethane (50 mL) wasthen added dropwise. The resulting solution was allowed to stir at −5°C. for three hours, and then allowed to warm to room temperature. Asolution of saturated aqueous sodium bicarbonate (200 mL) was thenadded. The organic layer was separated and the aqueous layer was washedwith dichloromethane (200 mL). The combined organic layers were washedwith a saturated aqueous sodium bicarbonate solution, dried over sodiumsulfate, filtered, and concentrated under reduced pressure. Theresulting residue was purified by silica gel column chromatography using80% hexanes/20% ethyl acetate to provide 11.43 g of a white solid. (88%yield) which was characterized by LC/MS (LCRS (MH) m/z: 386.42).

Boc-Tyr(Me)-OBn (2 g, 5.2 mmol) was dissolved in dichloromethane (15 mL)and cooled to 0° C. followed by dropwise addition of TFA (15 mL). Thereaction was allowed to warm to room temp and was stirred for 2 hours.The solvents were removed under reduced pressure to yield (AAA) as aclear oil (1.4 g, 95% yield) which was characterized by LC/MS (LCRS (MH)m/z: 286.42) and was used without further purification.

Synthesis of (BBB)

To a 0° C. solution of Boc-Ala-OH (750 mg, 3.9 mmol), H-Tyr(Me)-OBn (950mg, 3.3 mmol), HOBT (712 mg, 5.3 mmol) and HBTU (2.0 g, 5.3 mmol) inacetonitrile (60 mL) and DMF (6 mL) was added N,N-diisopropylethylamine(2.3 mL) dropwise. The mixture was stirred at 0° C. for 2 hours and wasthen diluted with ethyl acetate (300 mL) and washed with a saturatedaqueous sodium bicarbonate solution (2×100 mL) and brine (100 mL). Theorganic layers were dried over sodium sulfate, filtered and concentratedunder reduced pressure to yield an opaque oil that was purified bysilica gel column chromatography using 50% hexanes/50% ethyl acetate toyield 600 mg of (BBB) as a white foam (40% yield) that was characterizedby LC/MS (LCRS (MH) m/z: 457.52).

Synthesis of (CCC)

To a 0° C. solution of (BBB) (5.9 g, 12.9 mmol) in tetrahydrofuran (120mL) was added 10% Pd/C (1.2 g) and the resulting mixture was allowed tostir under 1 atmosphere of hydrogen for 2 hours. The mixture was thenfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The organic filtrate was then concentrated underreduced pressure and placed under high vacuum to provide 4.53 g (95%yield) of (CCC) that was used without further purification.

Synthesis of (DDD)

To a 0° C. solution of (CCC) (4 g, 10.9 mmol), 4 (2.23 g, 10.9 mmol),HOBT (2.36 g, 17.4 mmol) and HBTU (6.6 g, 17.4 mmol) in acetonitrile(200 mL) and DMF (5 mL) was added N,N-diisopropylethylamine (7.6 mL) andthe mixture was stirred at 0° C. for 2 hours. It was then diluted withethyl acetate (400 mL) and washed with saturated aqueous sodiumbicarbonate (2×100 mL) and brine (100 mL). The organic layers were driedover sodium sulfate, filtered, and concentrated under reduced pressure.The resulting residue was purified by HPLC (aqueous ammonium acetate(0.02 M) and acetonitrile) to provide (DDD) (4.47 g, 74% yield) ascharacterized by LC/MS (LCRS (MH) m/z: 554.79).

Synthesis of (EEE)

To a 0° C. solution of (DDD) (2 g, 3.6 mmol) in dichloromethane (32 mL)was added trifluoroacetic acid (8 mL), and the resulting solution wasstirred at that temperature for another hour. The solution was thenconcentrated under reduced pressure and placed under high vacuum toprovide (EEE) as confirmed by LC/MS (LCRS (MH) m/z: 454.72) that wasused without further purification.

Synthesis of 14

To a 0° C. solution of (EEE), morpholin-4-yl-acetic acid (1.048 g, 7.22mmol), HOBT (780 mg, 5.76 mmol) and HBTU (2.2 g, 5.76 mmol) inacetonitrile (60 mL) and DMF (3 mL) was added N,N-diisopropylethylamine(2.5 mL) dropwise. The mixture was stirred at 0° C. for 2 hours and wasthen diluted with ethyl acetate (300 mL) and washed with saturatedaqueous sodium bicarbonate (2×100 mL) and brine (100 mL). The organiclayers were dried over sodium sulfate, filtered, and concentrated underreduced pressure. The resulting residue was purified by HPLC (aqueousammonium acetate (0.02 M) and acetonitrile) to provide 14 (620 mg, 29%yield) which was characterized by LC/MS (LCRS (MH) m/z: 581.83).

To a 0° C. solution of (EEE) (65 mg, 0.144 mmol),(2R,6S)-2,6-dimethylmorpholin-4-yl)acetic acid hydrochloride (FFF) (50mg, 0.288 mmol), HOBT (32 mg, 0.23 mmol) and HBTU (88 mg, 0.23 mmol) inacetonitrile (15 mL) and DMF (1 mL), was added N,N-diisopropylethylamine(100 μL) dropwise and the mixture was stirred at 0° C. for 2 hours. Itwas then diluted with ethyl acetate (30 mL) and washed with saturatedaqueous sodium bicarbonate (2×15 mL) and brine (15 mL). The organiclayers were dried over sodium sulfate, filtered, and concentrated underreduced pressure to give a residue that was purified by HPLC (aqueousammonium acetate (0.02 M) and acetonitrile) to provide 15 (32 mg, 36%yield) as characterized by LC/MS (LCRS (MH) m/z: 609.83).

To a 0° C. solution of (EEE) (62 mg, 0.14 mmol),(2-methyl-1,3-thiazol-5-yl)acetic acid (GGG) (25 mg, 0.15 mmol), HOBT(30 mg, 0.22 mmol) and HBTU (84 mg, 0.22 mmol) in acetonitrile (15 mL)and DMF (1 mL) was added N,N-diisopropylethylamine (143 μL) dropwise andthe resulting mixture was stirred at 0° C. for 2 hours. It was thendiluted with ethyl acetate (30 mL) and washed with saturated aqueoussodium bicarbonate (2×15 mL) and brine (15 mL). The organic layers weredried over sodium sulfate, filtered, and concentrated under reducedpressure to give a residue that was purified by HPLC (aqueous ammoniumacetate (0.02 M) and acetonitrile) to provide 16 that was characterizedby LC/MS (LCRS (MH) m/z: 593.72).

Synthesis of (III)

To a 0° C. solution of (HHH) (2 g, 5.9 mmol), 4 (2.44 g, 11.89 mmol),HOBT (1.28 g, 9.5 mmol) and HBTU (3.6 g, 9.5 mmol) in acetonitrile (180mL) and DMF (10 mL) was added N,N-diisopropylethylamine (4.14 mL)dropwise and the mixture was stirred at 0° C. for 2 hours. It was thendiluted with ethyl acetate (200 mL) and washed with a saturated aqueoussodium bicarbonate solution (2×50 mL) and brine (50 mL). The combinedorganic layers were dried over sodium sulfate, filtered, andconcentrated under reduced pressure to give a residue that was purifiedby HPLC (aqueous ammonium acetate (0.02 M) and acetonitrile) to provide(III) (1.5 g, 50% yield) as characterized by LC/MS (LCRS (MH) m/z:524.71).

Synthesis of (JJJ)

To a 0° C. solution of (III) (60 mg, 0.1 mmol) in dichloromethane (2 mL)was added trifluoroacetic acid (0.5 mL), and the resulting solution wasstirred at that temperature for another hour. The solution was thenconcentrated under reduced pressure and placed under high vacuum toprovide (JJJ) as confirmed by LC/MS (LCRS (MH) m/z: 424.51) that wasused without further purification.

Synthesis of 17

To a 0° C. solution of (JJJ), (2,4-dimethyl-1,3-thiazol-5-yl-acetic acid(40 mg, 0.23 mmol), HOBT (25 mg, 0.183 mmol) and HBTU (70 mg, 0.183mmol) in acetonitrile (6 mL) and DMF (1 mL) was addedN,N-diisopropylethylamine (80 μL) dropwise. The mixture was then stirredat 0° C. for 2 hours. It was then diluted with ethyl acetate (50 mL) andwashed with saturated aqueous sodium bicarbonate (2×10 mL) and brine (10mL). The combined organic layers were dried over sodium sulfate,filtered, and concentrated under reduced pressure to give a residue thatwas purified by HPLC (aqueous ammonium acetate (0.02 M) andacetonitrile) to provide 17 (29 mg, 44% yield) which was characterizedby LC/MS (LCRS (MH) m/z: 577.86).

Example 18 Assays to Determine Inhibitory Preference

There are three types of assays that can be utilized when determiningwhether or not a molecule preferentially inhibits the CT-L activity ofthe constitutive or immunoproteasome. Enzyme kinetic assays such asthose disclosed in U.S. application Ser. No. 09/569,748, Example 2 andStein et al., Biochem. (1996), 35, 3899-3908 use isolated 20S proteasomepreps with greater than 90% constitutive proteasome subunits orimmunoproteasome subunits. The inhibitory preference of the molecule isthen based on the EC₅₀ ratio of the chymotryptic-like activity of theconstitutive proteasome to that of the immunoproteasome (20S ratio).

Alternatively, the CT-L EC₅₀ of a compound can be determined using 26Sproteasome in the context of a cell lysate. Compound is added to lysategenerated from cells that either predominantly express constitutiveproteasome (e.g., HT29) or immunoproteasome (e.g., THP1). Again, theinhibitory preference is then based on the EC₅₀ ratio (Lysate ratio).

Lastly, a more cell-based approach can be utilized. Cells expressingapproximately equivalent amounts of immuno- and constitutive proteasome(e.g., RPMI-8226) are treated with test compound, followed by the methodfor determining the activity of a proteasome inhibitor as described inapplication Ser. No. 11/254,541. The ratio of the EC₅₀ generated in theELISA-based assay using β5 antibody and LMP7 antibodies (ELISA ratio)provides the basis for determining the inhibitory preference of the testcompound. In all instances, a ratio of one indicates that the moleculeworks equally well at inhibiting the CT-L activity of both forms ofproteasome. In all three assays, a ratio of less than one denotes themolecule inhibits the CT-L activity of the constitutive proteasomebetter than that of the immunoproteasome. Ratios greater than onesignifies the molecule inhibits chymotrypsin-like activity of theimmunoproteasome better than that of the constitutive proteasome.

Example 19 ELISA Assay

A suitable ELISA assay may be found in U.S. patent application Ser. No.11/254,541, incorporated herein in its entirety. Briefly, RPMI-8226cells were treated with 0.1 nM to 1 μM of proteasome Inhibitor B. Thesamples were then washed with phosphate-buffered saline (PBS) and lysedin hypotonic buffer (20 mM Tris pH 8, 5 mM EDTA) (Tris-HCl and EDTA areavailable from Teknova, Inc., Hollister, Calif.). Cellular debris wasremoved by centrifugation at 14,000 rpm in a microfuge (4° C.) for 2min. The supernatant was transferred to a fresh tube, snap frozen inliquid nitrogen and stored at −80° C. After thawing on ice, the samples(30 μl for assays run in triplicate) were treated with 500 nM ofInhibitor A for 1 hr at room temperature. Following treatment withInhibitor A, the lysate was denatured by addition of seven volumes of 1%SDS (210 μl) (available from Bio-Rad, Hercules, Calif.) and heating at99° C. with vigorous shaking for 5 min. The sample was allowed to cooland two volumes (60 μl) of 10% Triton X-100 (available from Bio-Rad,Hercules, Calif.) was added.

Streptavidin sepharose beads (6.5 μl/well) (available from AmershamBiosciences, Piscataway, N.J.), were washed three times with 1 ml PBS(available from Mediatech, Inc., Herndon, Va.) in a microcentrifugetube. The beads were resuspended in ELISA wash/block buffer (PBS+0.1%Tween 20+1% bovine serum albumin; 20 μl/well) and transferred to thewells of a 96 well filter plate (BSA is available from Sigma, St. Louis,Mo.; Tween is available from Calbiochem, San Diego, Calif.). Denaturedwhole blood or PBMC lysates that were treated with Inhibitor 3 wereadded to the filter plate wells containing the streptavidin sepharosebeads (each sample assayed in triplicate) and incubated for 1 hr at roomtemperature with shaking (MultiScreen-DV Opaque Plates with low proteinbinding durapore membrane; available from Millipore, Billerica, Mass.).The unbound material was removed by gentle filtration and the beads werewashed six times with ELISA wash/block buffer (200 μl each).

Primary antibody to human 20S proteasome subunit β5 (rabbit polyclonalantibody; available from Biomol, Plymouth Meeting, Pa.) or human 20Simmunoproteasome subunit LMP7 (rabbit polyclonal antibody; availablefrom Affinity BioReagents, Golden, Colo.) was diluted 1:1000 in ELISAwash/block buffer, added to the beads (100 μl/well), and incubated for 1hr at room temperature on an orbital shaker. The beads were washed sixtimes with ELISA wash/block buffer with gentle filtration. Secondaryantibody treatment (1:5000) and washing are as described for primaryantibody (goat anti-rabbit antibody-HRP conjugate; available fromBiosource, Camarillo, Calif.). The beads were then resuspended in 100 μlchemiluminescent detection reagent (Super Signal Pico ChemiluminescentSubstrate™; available from Pierce, Rockford, Ill.) and luminescence wasread on a Tecan plate reader.

Occupation of the active sites of the proteasome with the peptideepoxyketone inhibitor results in both a decrease in chymotryptic-likecatalytic activity and a decrease in binding of the biotinylated probe(Inhibitor A). These data suggest that the ELISA-based assay using thebiontinylated probe accurately reflects the inhibitory activity ofInhibitor B.

An exemplary feature of the ELISA-based PD assay is that it permitsdifferentiation between constitutive proteasome inhibition (β5) andimmunoproteasome inhibition (LMP7) because it utilizes subunit-specificantibodies.

Utilizing a different active site probe (Inhibitor C) expands theutility of the ELISA-based assay for measuring the occupation ofmultiple constitutive (β5, β1, β2) and immunoproteasome (LMP7, LMP2)active sites in 8226 multiple myeloma cell line that co-expresses bothforms of proteasome. The expanded active site assay can be used tomeasure relative inhibitor selectivity both between the immuno- andconstitutive proteasomes as well as among the three active sites of eachproteasome. In addition, the ELISA-based assay is able to determine thepotency and selectivity of other classes of proteasome inhibitors,including the peptide boronic acid-based inhibitors.

To conduct a pharmacodynamic evaluation of an inhibitor, whole blood andPBMC samples are collected prior to dosing and at multiple time-pointsafter dosing. Lysates are prepared and protein concentration assaysperformed to normalize for total protein in each lysate. The level ofinhibitor binding to β5 and LMP7 subunits in whole blood andPBMCRPMI-8226 cells, respectively, is determined by thestreptavidin-capture ELISA described above. Standard curves withpurified constitutive proteasome and immunoproteasome are utilized toensure assay linearity/dynamic range and to convert thechemiluminescence signal to an absolute amount (μg) of subunit bound.The ratio of the EC50 generated in the ELISA-based assay using β5antibody and LMP7 antibodies (ELISA ratio) provides the basis fordetermining the inhibitory preference of the test compound. The aboveinhibitor (B) has a ratio greater than 20, thus, it is much moreselective at inhibiting the chymotryptic-like activity associated withthe immunoproteasome.

To determine the level of proteasome inhibition for a given patient, theamount of β5 or LMP7 detected in the post-dose sample is compared to thepre-dose sample. Proteasome inhibition is determined after a single doseor after a cycle of dosing, or is used to monitor inhibition shortlyafter dosing as well as to monitor recovery of proteasome activity aftera course of dosing.

Example 20 Biological Results

20S ratio Lysate ratio (const: (HT29: Elisa ratio Structure immuno)Sultan/Thp1) (beta5:LMP7)

>1.0 >3.0 >1.0

>2.0 >2.0 >3.0

<1.0 <1.0 <1.0

>2.0 >3.0 >5.0

>1.0 >2.0 >5.0

<0.5 <0.5 <0.5

<0.5

>1.0 >5.0 >1.0

>1.0 >3.0 >2.0

>1.0 >1.0 >3.0

>2.0 >5.0 >3.0

>5.0 >3.0 >3.0

<0.5 <1.0 <0.5

>3.0 >5.0 >2.0

>5.0 >3.0 >2.0

>5.0 >5.0 >3.0

<0.5

<1.0 <1.0 <0.5

>3.0 >3.0 >2.0

>5.0 >2.0 >5.0

>5.0 >3.0 >5.0

>5.0 >3.0 >5.0

<0.5 <0.5 <0.5

<0.5 <0.5 <1.0

<0.5

<0.5

<0.5 <0.5 <1.0

>5.0 >5.0 >5.0

>3.0 >3.0 >1.0

>2.0 >5.0 >5.0

<0.5

<0.5 <0.5 <0.5

>5.0 >5.0 >3.0

>5.0 >2.0 >3.0

>5.0 >3.0 >5.0

<0.5 <0.5 <0.5

>1.0 >2.0 >5.0

>2.0 >2.0 >3.0

>5.0 >3.0 >5.0

>5.0 >5.0 >5.0

>2.0 >2.0 >5.0

<1.0

>3.0 >3.0 >2.0

>5.0 >2.0 >2.0

>5.0 >2.0 >1.0

>5.0 >5.0 >5.0

<0.5

>2.0 >3.0 >3.0

>1.0 >5.0 >1.0

>1.0 >2.0 >5.0

<0.5 <0.5 <0.5

>2.0 >2.0 >3.0

>3.0 >3.0 >5.0

>2.0 >3.0 >1.0

>1.0 >1.0 >3.0

Example 21 Use of Immunoproteasome Inhibitor in Rheumatoid ArthritisModel

The effect of Compound 14 on disease progression was assessed in 2 mousemodels of rheumatoid arthritis (FIG. 2). In the arthrogenic antibodymodel, in which disease is induced by the administration ofanti-collagen antibodies and lipopolysaccharide (LPS) (Terato et al., JImmunol 148:2103-2108, 1992), Compound 14 inhibited disease progressionin a dose dependent manner (FIG. 2A). Rheumatoid arthritis was inducedon Day 0 in female Balb/c mice by IV administration of anti-type IIcollagen antibodies followed 3 days later by LPS. Compound X wasadministered IV 3 times/week for 2 weeks beginning on Day 4, the firstday animals showed evidence of disease. Per mouse, each paw was measuredfor disease using a scale of 0-4 and a total clinical score was assignedfor each animal (max score=16). Administration of 6 mg/kg of Compound 14reduced disease severity by ˜50% while the 20 mg/kg dose level inhibitedthe disease by greater than 75%.

The effect administration of Compound 14 on disease progress was alsoassessed in an alternative mouse model for RA, in which disease develops21-30 days after immunization with bovine type II collagen (Kagari etal., J Immunol 169:1459-1466, 2002). Administration of 6 or 20 mg/kgCompound 14 beginning after first signs of disease inhibited diseaseprogression as compared to vehicle control (FIG. 2 B). Again, diseaseprogression was measured using a total clinical score of paw conditionper mouse. As seen previously, increasing amounts of Compound 14resulted in enhanced reduction of disease severity.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

We claim:
 1. A method for the treatment of an immune-related disease ina patient and said patient suffers from an immune-related diseaseselected from the group consisting of myositis, atopic dermatitis,scleroderma, ankylosing spondylitis, dermatomyositis, and chronicgranulomatous, comprising administering to the patient a compound havinga structure of formula (I) or a pharmaceutically acceptable saltthereof,

wherein each A is independently selected from C═O, C═S, and SO₂; or A isoptionally a covalent bond when adjacent to an occurrence of Z; B isabsent or is N(R⁹)R¹⁰; L is absent or is selected from C═O, C═S, andSO₂; M is C₁₋₁₂alkyl; Q is absent or is selected from O, NH, andN—C₁₋₆alkyl; X is selected from O, S, NH, and N—C₁₋₆alkyl; each Z isindependently selected from O, S, NH, and N—C₁₋₆alkyl; or Z isoptionally a covalent bond when adjacent to an occurrence of A; R¹ isselected from H, —C₁₋₆alkyl-B; C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,and C₁₋₆aralkyl; R² and R³ are each independently selected from aryl,C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl; R⁴ is N(R⁵)L-Q-R⁶; R⁵ isselected from hydrogen, OH, aryl C₁₋₆alkyl, and C₁₋₆alkyl; R⁶ isselected from an N-terminal protecting group,hetoercyclylMZAZC₁₋₆alkyl-, heterocycylM-, and carbocycylM; R⁷ and R⁸are independently selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl; R⁹is selected from hydrogen, OH, and C₁₋₆aklyl; R¹⁰ is an N-terminalprotecting group; and R¹⁵ is selected from C₁₋₆alkyl andC₁₋₆hydroxyalkyl; provided that in any occurrence of the sequence ZAZ,at least one member of the sequence must be other than a covalent bond.2. The method of claim 1, wherein the compound of formula (I) has astructure:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein L is C═O, Q is absent, M is C₁₋₈alkyl and R⁶ is heterocyclylM-and the heterocyclyl moiety is morpholino.
 4. A method of treating apatient suffering from a disease selected from the group consisting ofdiabetic nephropathy, glomerulosclerosis, congestive heart failure, andscleroderma, comprising administering to the patient a compound having astructure of formula (I) or a pharmaceutically acceptable salt thereof,

wherein each A is independently selected from C═O, C═S, and SO₂; or A isoptionally a covalent bond when adjacent to an occurrence of Z; B isabsent or is N(R⁹)R¹⁰; L is absent or is selected from C═O, C═S, andSO₂; M is C₁₋₁₂alkyl; Q is absent or is selected from 0, NH, andN—C₁₋₆alkyl; X is selected from O, S, NH, and N—C₁₋₆alkyl; each Z isindependently selected from O, S, NH, and N—C₁₋₆alkyl; or Z isoptionally a covalent bond when adjacent to an occurrence of A; R¹ isselected from H, —C₁₋₆alkyl-B; C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,and C₁₋₆aralkyl; R² and R³ are each independently selected from aryl,C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl; R⁴ is N(R⁵)L-Q-R⁶; R⁵ isselected from hydrogen, OH, aryl C₁₋₆alkyl, and C₁₋₆alkyl; R⁶ isselected from an N-terminal protecting group,hetoercyclylMZAZC₁₋₆alkyl-, heterocycylM-, and carbocycylM; R⁷ and R⁸are independently selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl; R⁹is selected from hydrogen, OH, and C₁₋₆aklyl; R¹⁰ is an N-terminalprotecting group; and R¹⁵ is selected from C₁₋₆alkyl andC₁₋₆hydroxyalkyl; provided that in any occurrence of the sequence ZAZ,at least one member of the sequence must be other than a covalent bond.5. The method of claim 4, wherein the compound of formula (I) has astructure:

or a pharmaceutically acceptable salt thereof.
 6. The method of claim 4,wherein L is C═O, Q is absent, M is C₁₋₈alkyl and R⁶ is heterocyclylM-and the heterocyclyl moiety is morpholino.
 7. A method of treatingstroke in a patient, comprising administering to the patient a compoundhaving a structure of formula (I) or a pharmaceutically acceptable saltthereof,

wherein each A is independently selected from C═O, C═S, and SO₂; or A isoptionally a covalent bond when adjacent to an occurrence of Z; B isabsent or is N(R⁹)R¹⁰; L is absent or is selected from C═O, C═S, andSO₂; M is C₁₋₁₂alkyl; Q is absent or is selected from O, NH, andN—C₁₋₆alkyl; X is selected from O, S, NH, and N—C₁₋₆alkyl; each Z isindependently selected from O, S, NH, and N—C₁₋₆alkyl; or Z isoptionally a covalent bond when adjacent to an occurrence of A; R¹ isselected from H, —C₁₋₆alkyl-B; C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,and C₁₋₆aralkyl; R² and R³ are each independently selected from aryl,C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl; R⁴ is N(R⁵)L-Q-R⁶; R⁵ isselected from hydrogen, OH, aryl C₁₋₆alkyl, and C₁₋₆alkyl; R⁶ isselected from an N-terminal protecting group,hetoercyclylMZAZC₁₋₆alkyl-, heterocycylM-, and carbocycylM; R⁷ and R⁸are independently selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl; R⁹is selected from hydrogen, OH, and C₁₋₆aklyl; R¹⁰ is an N-terminalprotecting group; and R¹⁵ is selected from C₁₋₆alkyl andC₁₋₆hydroxyalkyl; provided that in any occurrence of the sequence ZAZ,at least one member of the sequence must be other than a covalent bond.8. The method of claim 7, wherein the compound of formula (I) has astructure:

or a pharmaceutically acceptable salt thereof.
 9. The method of claim 7,wherein L is C═O, Q is absent, M is C₁₋₈alkyl and R⁶ is heterocyclylM-and the heterocyclyl moiety is morpholino.
 10. A method of treating apatient suffering from inflammation or an inflammatory disease,comprising administering to the patient a compound having a structure offormula (I) or a pharmaceutically acceptable salt thereof,

wherein each A is independently selected from C═O, C═S, and SO₂; or A isoptionally a covalent bond when adjacent to an occurrence of Z; B isabsent or is N(R⁹)R¹⁰; L is absent or is selected from C═O, C═S, andSO₂; M is C₁₋₁₂alkyl; Q is absent or is selected from O, NH, andN—C₁₋₆alkyl; X is selected from O, S, NH, and N—C₁₋₆alkyl; each Z isindependently selected from O, S, NH, and N—C₁₋₆alkyl; or Z isoptionally a covalent bond when adjacent to an occurrence of A; R¹ isselected from H, —C₁₋₆alkyl-B; C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,and C₁₋₆aralkyl; R² and R³ are each independently selected from aryl,C₁₋₆aralkyl, heteroaryl, and C₁₋₆heteroaralkyl; R⁴ is N(R⁵)L-Q-R⁶; R⁵ isselected from hydrogen, OH, aryl C₁₋₆alkyl, and C₁₋₆alkyl; R⁶ isselected from an N-terminal protecting group,hetoercyclylMZAZC₁₋₆alkyl-, heterocycylM-, and carbocycylM; R⁷ and R⁸are independently selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl; R⁹is selected from hydrogen, OH, and C₁₋₆aklyl; R¹⁰ is an N-terminalprotecting group; and R¹⁵ is selected from C₁₋₆alkyl andC₁₋₆hydroxyalkyl; provided that in any occurrence of the sequence ZAZ,at least one member of the sequence must be other than a covalent bond.11. The method of claim 10, wherein the compound of formula (I) has astructure:

or a pharmaceutically acceptable salt thereof.
 12. The method of claim10, wherein L is C═O, Q is absent, M is C₁₋₈alkyl and R⁶ isheterocyclylM- and the heterocyclyl moiety is morpholino.
 13. The methodof claim 10, wherein the inflammatory disease is selected from the groupconsisting of bronchitis, conjunctivitis, pancreatitis, chroniccholecystitis, bronchiectasis, aortic valve stenosis, restenosis,psoriatic arthritis, fibrosis, infection, and ischemia.
 14. The methodof claim 10, wherein the inflammatory disease is selected from the groupconsisting of lupus, rheumatoid arthritis, scleroderma, ankylosingspondylitis, dermatomyositis, psoriasis, multiple sclerosis andinflammatory bowel disease.
 15. The method of claim 14, wherein theinflammatory bowel disease is ulcerative colitis or Crohn's disease.